Structured Review

Bio-Rad electroporation cuvettes
A , Iron incorporation as quantified by the ferrozin assay. No increase in intracellular iron was detected following <t>electroporation</t> with Sinerem. MEP with Supravist at a concentration of 3 mg Fe/mL resulted in a 400% increase in intracellular iron concentration.
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Images

1) Product Images from "Magnetic Resonance Imaging of Monocytes Labeled with Ultrasmall Superparamagnetic Particles of Iron Oxide Using Magnetoelectroporation in an Animal Model of Multiple Sclerosis"

Article Title: Magnetic Resonance Imaging of Monocytes Labeled with Ultrasmall Superparamagnetic Particles of Iron Oxide Using Magnetoelectroporation in an Animal Model of Multiple Sclerosis

Journal:

doi:

A , Iron incorporation as quantified by the ferrozin assay. No increase in intracellular iron was detected following electroporation with Sinerem. MEP with Supravist at a concentration of 3 mg Fe/mL resulted in a 400% increase in intracellular iron concentration.
Figure Legend Snippet: A , Iron incorporation as quantified by the ferrozin assay. No increase in intracellular iron was detected following electroporation with Sinerem. MEP with Supravist at a concentration of 3 mg Fe/mL resulted in a 400% increase in intracellular iron concentration.

Techniques Used: Electroporation, Concentration Assay

2) Product Images from "Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli"

Article Title: Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-4-11

Time course for promotion of hyper-rec phenotype. EHEC strain TUV93-0 containing pKM208 (five cultures, 20 ml each) was grown for electrocompetence as described in the Methods section. At various times prior to collection, IPTG was added to four of the cultures to a final concentration of 1 mM; the fifth culture received no IPTG. The cells were heat shocked for the final 15 minutes, prepared for electroporation and electroporated with DNA (~0.25 μg) containing the kan gene flanked by 40 bp of EHEC DNA (resulting in a deletion of O-islands #130 and #131). After suspension in LB, the cells were grown for 90 minutes at 37°C and plated on LB plates containing 20 μg/ml kanamycin. The number of Kan R transformants per 10 8 survivor and total number of Kan R transformants are plotted as a function of IPTG concentration. The data points are averages of two experiments (ranges are shown). A random check of 160 colonies showed that 95% re-struck successfully to fresh LB plates containing 20 μg/ml kanamycin; 10 of 10 of these colonies were verified by PCR analysis to be true recombinants (data not shown). Insert: 0.1 ml of electrocompetent cells, prepared with and without 1 hour IPTG induction, were spread on LB plates containing 100 μg/ml rifampicin to determine total number of Rif R mutants. Dilutions of the cells were titered on LB plates to determine total cell density; experiments done in triplicate (+/- standard error).
Figure Legend Snippet: Time course for promotion of hyper-rec phenotype. EHEC strain TUV93-0 containing pKM208 (five cultures, 20 ml each) was grown for electrocompetence as described in the Methods section. At various times prior to collection, IPTG was added to four of the cultures to a final concentration of 1 mM; the fifth culture received no IPTG. The cells were heat shocked for the final 15 minutes, prepared for electroporation and electroporated with DNA (~0.25 μg) containing the kan gene flanked by 40 bp of EHEC DNA (resulting in a deletion of O-islands #130 and #131). After suspension in LB, the cells were grown for 90 minutes at 37°C and plated on LB plates containing 20 μg/ml kanamycin. The number of Kan R transformants per 10 8 survivor and total number of Kan R transformants are plotted as a function of IPTG concentration. The data points are averages of two experiments (ranges are shown). A random check of 160 colonies showed that 95% re-struck successfully to fresh LB plates containing 20 μg/ml kanamycin; 10 of 10 of these colonies were verified by PCR analysis to be true recombinants (data not shown). Insert: 0.1 ml of electrocompetent cells, prepared with and without 1 hour IPTG induction, were spread on LB plates containing 100 μg/ml rifampicin to determine total number of Rif R mutants. Dilutions of the cells were titered on LB plates to determine total cell density; experiments done in triplicate (+/- standard error).

Techniques Used: Concentration Assay, Electroporation, Polymerase Chain Reaction

3) Product Images from "Nucleolin-targeted Extracellular Vesicles as a Versatile Platform for Biologics Delivery to Breast Cancer"

Article Title: Nucleolin-targeted Extracellular Vesicles as a Versatile Platform for Biologics Delivery to Breast Cancer

Journal: Theranostics

doi: 10.7150/thno.16532

Characterization of AS1411-EVs for breast cancer targeting: A. Zeta potential distribution of EVs (blue), AS1411-EVs (red) and AS1411-EVs digested by DNase I (green) measured by nanoparticle tracking analysis. B. Fluorescence microscopy analysis of the T-AS1411 stable binding with EVs membrane outer surface. T-AS1411 was modified by Cy3 and bound to EVs via cholesterol affinity. The Cy3-AS1411-EVs were digested by DNase I and then captured by Dynabeads ® with CD63 antibody (down). The resulting fluorescence signal was very weak. However, control group Cy3-AS1411-EVs not digested by DNase I (up) had a very strong fluorescence signal. (BL: bright light, FL: fluorescence light, Scale bars: 100 μm). C. Size distribution of AS1411-EVs measured by nanoparticle tracking analysis. The peak diameter was at 77 nm for AS1411-EVs. D. AS1411-EVs-let-7-Cy3 was captured by beads and the fluorescence signal detected by fluorescence microscopy. The red fluorescence signal showed successful electroporation of let-7-Cy3 into EVs (Scale bars: 100 μm). E. Transmission electron microscopy of AS1411-EVs-let-7. The morphology of AS1411-EVs-let-7 was intact and the size was approximately 30-100 nm (Arrows indicate AS1411-EVs-let-7, Scale bars: 100 nm). F. Release kinetics of encapsulated cel-miRNA-67 from the AS1411-EVs as determined by incubation for up to 24 hours in PBS or mouse serum at 37°C (n=3) (Each bar represents the mean ±SD of three replicates).
Figure Legend Snippet: Characterization of AS1411-EVs for breast cancer targeting: A. Zeta potential distribution of EVs (blue), AS1411-EVs (red) and AS1411-EVs digested by DNase I (green) measured by nanoparticle tracking analysis. B. Fluorescence microscopy analysis of the T-AS1411 stable binding with EVs membrane outer surface. T-AS1411 was modified by Cy3 and bound to EVs via cholesterol affinity. The Cy3-AS1411-EVs were digested by DNase I and then captured by Dynabeads ® with CD63 antibody (down). The resulting fluorescence signal was very weak. However, control group Cy3-AS1411-EVs not digested by DNase I (up) had a very strong fluorescence signal. (BL: bright light, FL: fluorescence light, Scale bars: 100 μm). C. Size distribution of AS1411-EVs measured by nanoparticle tracking analysis. The peak diameter was at 77 nm for AS1411-EVs. D. AS1411-EVs-let-7-Cy3 was captured by beads and the fluorescence signal detected by fluorescence microscopy. The red fluorescence signal showed successful electroporation of let-7-Cy3 into EVs (Scale bars: 100 μm). E. Transmission electron microscopy of AS1411-EVs-let-7. The morphology of AS1411-EVs-let-7 was intact and the size was approximately 30-100 nm (Arrows indicate AS1411-EVs-let-7, Scale bars: 100 nm). F. Release kinetics of encapsulated cel-miRNA-67 from the AS1411-EVs as determined by incubation for up to 24 hours in PBS or mouse serum at 37°C (n=3) (Each bar represents the mean ±SD of three replicates).

Techniques Used: Fluorescence, Microscopy, Binding Assay, Modification, Electroporation, Transmission Assay, Electron Microscopy, Incubation

4) Product Images from "A Synthetic Disaccharide Derivative of Diphyllin, TAARD, Activates Human Natural Killer Cells to Secrete Interferon-Gamma via Toll-Like Receptor-Mediated NF-κB and STAT3 Signaling Pathways"

Article Title: A Synthetic Disaccharide Derivative of Diphyllin, TAARD, Activates Human Natural Killer Cells to Secrete Interferon-Gamma via Toll-Like Receptor-Mediated NF-κB and STAT3 Signaling Pathways

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01509

TAARD increases the luciferase reporter activities of NF-κB and STAT3 via TLR1 and TLR3, respectively. (A) NKL cells were electroporated with either pGL3-κB-Luc (1 µg) or pGL3-Basic (1 µg) and a pRL-TK renilla-luciferase control plasmid (50 ng). After electroporation, cells were immediately transferred into fresh medium and cultured for an additional 6 h. Then, cells were treated with different concentrations of TAARD. (B) NKL cells were electroporated as described in (A) but with 4×M67 pTATA TK-Luc plasmid (1 µg). Cells were treated and measured as described in (A) . (C) 293T cells were co-transfected with pGL3-κB-luc (1 µg) or pGL3-Basic (1 µg) and pRL-TK renilla-luciferase control plasmids (5 ng) in the presence or absence of a TLR1 (0.5 µg) expression plasmid by Lipofectamine 2000. Twenty-four hours later, cells were treated with different concentrations (0.1, 1, and 10 µM) of TAARD for another 24 h with fresh medium. (D) 293T cells were co-transfected with 4×M67 pTATA TK-Luc (1 µg) or pGL Basic plasmid (1 µg) and pRL-TK renilla-luciferase control plasmids (5 ng) in the presence or absence of a TLR3 (0.5 µg) expression plasmid by Lipofectamine 2000. Twenty-four hours later, cells were treated with different concentrations (0.1, 1, and 10 µM) of TAARD for another 24 h with fresh medium. The ratio of firefly to renilla luciferase activities was used to show the relative luciferase activity, which corresponded to NF-κB or STAT activation. Data shown represent one of three independent experiments with similar results. * p
Figure Legend Snippet: TAARD increases the luciferase reporter activities of NF-κB and STAT3 via TLR1 and TLR3, respectively. (A) NKL cells were electroporated with either pGL3-κB-Luc (1 µg) or pGL3-Basic (1 µg) and a pRL-TK renilla-luciferase control plasmid (50 ng). After electroporation, cells were immediately transferred into fresh medium and cultured for an additional 6 h. Then, cells were treated with different concentrations of TAARD. (B) NKL cells were electroporated as described in (A) but with 4×M67 pTATA TK-Luc plasmid (1 µg). Cells were treated and measured as described in (A) . (C) 293T cells were co-transfected with pGL3-κB-luc (1 µg) or pGL3-Basic (1 µg) and pRL-TK renilla-luciferase control plasmids (5 ng) in the presence or absence of a TLR1 (0.5 µg) expression plasmid by Lipofectamine 2000. Twenty-four hours later, cells were treated with different concentrations (0.1, 1, and 10 µM) of TAARD for another 24 h with fresh medium. (D) 293T cells were co-transfected with 4×M67 pTATA TK-Luc (1 µg) or pGL Basic plasmid (1 µg) and pRL-TK renilla-luciferase control plasmids (5 ng) in the presence or absence of a TLR3 (0.5 µg) expression plasmid by Lipofectamine 2000. Twenty-four hours later, cells were treated with different concentrations (0.1, 1, and 10 µM) of TAARD for another 24 h with fresh medium. The ratio of firefly to renilla luciferase activities was used to show the relative luciferase activity, which corresponded to NF-κB or STAT activation. Data shown represent one of three independent experiments with similar results. * p

Techniques Used: Luciferase, Plasmid Preparation, Electroporation, Cell Culture, Transfection, Expressing, Activity Assay, Activation Assay

5) Product Images from "The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation"

Article Title: The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation

Journal: Toxins

doi: 10.3390/toxins8040090

Effect of TcsL-cat or TcsL mutants treatment on cell viability. Cells were electroporated with TcsL-cat or mutants and proliferation tests were performed 72 h after electroporation. Data are expressed as mean ± SEM of at least three independent experiments. Statistically significant differences between results were evaluated by t test with Welch correction. A p value of
Figure Legend Snippet: Effect of TcsL-cat or TcsL mutants treatment on cell viability. Cells were electroporated with TcsL-cat or mutants and proliferation tests were performed 72 h after electroporation. Data are expressed as mean ± SEM of at least three independent experiments. Statistically significant differences between results were evaluated by t test with Welch correction. A p value of

Techniques Used: Electroporation

6) Product Images from "MR Imaging of Transplanted Stem Cells in Myocardial Infarction"

Article Title: MR Imaging of Transplanted Stem Cells in Myocardial Infarction

Journal:

doi: 10.1007/978-1-60761-901-7_10

Instant MR labeling of cells using magnetoelectroporation (MEP). a Cells are suspended in a sterile electroporation cuvette ( arrow ), mixed with Feridex ® , and placed in a cuvette holder. b Using the connected electroporator, cells are mildly permeabilized
Figure Legend Snippet: Instant MR labeling of cells using magnetoelectroporation (MEP). a Cells are suspended in a sterile electroporation cuvette ( arrow ), mixed with Feridex ® , and placed in a cuvette holder. b Using the connected electroporator, cells are mildly permeabilized

Techniques Used: Labeling, Electroporation

7) Product Images from "The recurrent SET-NUP214 fusion as a new HOXA activation mechanism in pediatric T-cell acute lymphoblastic leukemia"

Article Title: The recurrent SET-NUP214 fusion as a new HOXA activation mechanism in pediatric T-cell acute lymphoblastic leukemia

Journal:

doi: 10.1182/blood-2007-09-111872

siRNA knockdown of SET and SET-NUP214 in the LOUCY T-ALL cell line. (A) SET expression using Western blot analysis 4 days after electroporation with the following conditions: no siRNA, control siRNA, siRNA SET exon 8, or siRNA SET exon 5. (B) Relative
Figure Legend Snippet: siRNA knockdown of SET and SET-NUP214 in the LOUCY T-ALL cell line. (A) SET expression using Western blot analysis 4 days after electroporation with the following conditions: no siRNA, control siRNA, siRNA SET exon 8, or siRNA SET exon 5. (B) Relative

Techniques Used: Expressing, Western Blot, Electroporation

ChIP and coIP analysis of T-ALL cell lines LOUCY and SKW3. (A) ChIP analysis of SKW3, LOUCY, and LOUCY 4 days after electroporation with siRNA SET exon 5, for promoter sequences of HOXA1 , HOXA3 , HOXA9 , HOXA10 , and HOXA11 . The amount of bound DNA was calculated
Figure Legend Snippet: ChIP and coIP analysis of T-ALL cell lines LOUCY and SKW3. (A) ChIP analysis of SKW3, LOUCY, and LOUCY 4 days after electroporation with siRNA SET exon 5, for promoter sequences of HOXA1 , HOXA3 , HOXA9 , HOXA10 , and HOXA11 . The amount of bound DNA was calculated

Techniques Used: Chromatin Immunoprecipitation, Co-Immunoprecipitation Assay, Electroporation

8) Product Images from "Hydra Mesoglea Proteome Identifies Thrombospondin as a Conserved Component Active in Head Organizer Restriction"

Article Title: Hydra Mesoglea Proteome Identifies Thrombospondin as a Conserved Component Active in Head Organizer Restriction

Journal: Scientific Reports

doi: 10.1038/s41598-018-30035-2

β-Catenin-dependent regulation of HmTSP expression. ( a ) Topography of the HmTSP promoter including -2191 bp of 5′ untranslated region. Black boxes depict the first two exons of the HmTSP gene. The arrow indicates the transcription start site of the HmTSP mRNA (accession no. XM_012702849), and ATG the translation start site. Positions of canonical TCF binding motifs are indicated by red (5′-CTTTGTT-3′) or blue (5′-AACAAAG-3′) bars, respectively. The localization and size of DNA segments flanked by specific ChIP primer pairs are indicated by horizontal grey bars. ( b ) ChIP analysis of the HmTSP promoter region using chromatin from whole Hydra animals. A polyclonal antibody directed against Hydra TCF was used for immunoprecipitation, followed by PCR amplification of the indicated fragments from the HmTSP regulatory region including a region without a TCF binding site (control). Reactions with normal rabbit serum (NRS) or total chromatin (Input) were used as further controls. PCR products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. One representative of two replicates using independent chromatin preparations is shown (see Supplementary Fig. S3a ). ( c , d ) ISH analysis of HmTSP expression (red) in the head regions of transgenic animals with ectodermal GFP expression (green) electroporated with control GFP siRNA ( c ) or GFP and β-Catenin siRNAs ( d ). HmTSP transcript was absent in electroporated areas with impaired β-Catenin expression. Note that GFP-positive areas indicate patches of tissue not affected by the electroporation. ISH was performed 8 days after electroporation with siRNAs. Scale bars = 50 µm. Representative of 10 hydras examined. E . Quantitative rt PCR analysis of β-Catenin and HmTSP expression upon siRNA mediated β-Catenin knockdown. Reduced expression of β-Catenin in siRNA-treated animals correlated with a major decrease in expression of HmTSP . Columns represent the mean and error bars the standard deviation from three independent experiments.
Figure Legend Snippet: β-Catenin-dependent regulation of HmTSP expression. ( a ) Topography of the HmTSP promoter including -2191 bp of 5′ untranslated region. Black boxes depict the first two exons of the HmTSP gene. The arrow indicates the transcription start site of the HmTSP mRNA (accession no. XM_012702849), and ATG the translation start site. Positions of canonical TCF binding motifs are indicated by red (5′-CTTTGTT-3′) or blue (5′-AACAAAG-3′) bars, respectively. The localization and size of DNA segments flanked by specific ChIP primer pairs are indicated by horizontal grey bars. ( b ) ChIP analysis of the HmTSP promoter region using chromatin from whole Hydra animals. A polyclonal antibody directed against Hydra TCF was used for immunoprecipitation, followed by PCR amplification of the indicated fragments from the HmTSP regulatory region including a region without a TCF binding site (control). Reactions with normal rabbit serum (NRS) or total chromatin (Input) were used as further controls. PCR products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. One representative of two replicates using independent chromatin preparations is shown (see Supplementary Fig. S3a ). ( c , d ) ISH analysis of HmTSP expression (red) in the head regions of transgenic animals with ectodermal GFP expression (green) electroporated with control GFP siRNA ( c ) or GFP and β-Catenin siRNAs ( d ). HmTSP transcript was absent in electroporated areas with impaired β-Catenin expression. Note that GFP-positive areas indicate patches of tissue not affected by the electroporation. ISH was performed 8 days after electroporation with siRNAs. Scale bars = 50 µm. Representative of 10 hydras examined. E . Quantitative rt PCR analysis of β-Catenin and HmTSP expression upon siRNA mediated β-Catenin knockdown. Reduced expression of β-Catenin in siRNA-treated animals correlated with a major decrease in expression of HmTSP . Columns represent the mean and error bars the standard deviation from three independent experiments.

Techniques Used: Expressing, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, In Situ Hybridization, Transgenic Assay, Electroporation, Quantitative RT-PCR, Standard Deviation

Functional analysis of HmTSP by siRNA knockdown. ( a ) ISH analysis of HmTSP expression in control GFP -transgenic animals electroporated with GFP siRNA. ( b ) ISH analysis showing reduced HmTSP expression in animals electroporated with HmTSP siRNA. Bars = 100 µm. ( c , d ) Demonstration of effective siRNA knockdown for GFP in transgenic hydra with ectodermal GFP expression. ( c ) Untreated control animal. D. Representative animal of the same transgenic strain as in C 8 days after electroporation with GFP siRNA. Bars = 500 µm. ( e– e’) Treatment with combined siGFP and siHmTSP does not induce morphological changes in steady state polyps. ( f , g ) Treatment with ALP of siTSP electroporated animals resulted in a dramatic increase of ectopic tentacles compared to the siGFP-treated control group. f’ and g’ show the reduced GFP expression in the respective animals. Each panel representative of at least 10 hydras examined. ( h ) Quantification of ALP-induced ectopic tentacles in animals electroporated with siGFP or siGFP and siTSP. Animals electroporated with the respective siRNAs without subsequent ALP treatment served as controls. ALP treatment was performed 8 days after electroporation and the numbers of tentacles/animal in each group were counted 5 days after ALP treatment. Animals in d and e were recorded at the same time point after electroporation as animals in f and g. Animals (n) in each group were: siGFP = 67; siGFP/siTSP = 67; siGFP/ALP = 71; siGFP/siTSP/ALP = 71. ( i ) Effect of Wnt3 depletion on ectopic tentacle formation: Animals were electroporated with siRNAs specific for GFP or directed against Wnt3 and GFP followed by ALP- or control treatment as indicated. Ectopic tentacle formation was analyzed as described above ( h ). Number of polyps analyzed in each group was: siGFP: 74, siGFP/siWnt3: 76, siGFP/ALP: 79, and siGFP/siWnt3/ALP: 81. ( h and i show the results from three independent experiments. Each data point represents a single hydra, bars indicate the mean ± S.E.M. ***P value
Figure Legend Snippet: Functional analysis of HmTSP by siRNA knockdown. ( a ) ISH analysis of HmTSP expression in control GFP -transgenic animals electroporated with GFP siRNA. ( b ) ISH analysis showing reduced HmTSP expression in animals electroporated with HmTSP siRNA. Bars = 100 µm. ( c , d ) Demonstration of effective siRNA knockdown for GFP in transgenic hydra with ectodermal GFP expression. ( c ) Untreated control animal. D. Representative animal of the same transgenic strain as in C 8 days after electroporation with GFP siRNA. Bars = 500 µm. ( e– e’) Treatment with combined siGFP and siHmTSP does not induce morphological changes in steady state polyps. ( f , g ) Treatment with ALP of siTSP electroporated animals resulted in a dramatic increase of ectopic tentacles compared to the siGFP-treated control group. f’ and g’ show the reduced GFP expression in the respective animals. Each panel representative of at least 10 hydras examined. ( h ) Quantification of ALP-induced ectopic tentacles in animals electroporated with siGFP or siGFP and siTSP. Animals electroporated with the respective siRNAs without subsequent ALP treatment served as controls. ALP treatment was performed 8 days after electroporation and the numbers of tentacles/animal in each group were counted 5 days after ALP treatment. Animals in d and e were recorded at the same time point after electroporation as animals in f and g. Animals (n) in each group were: siGFP = 67; siGFP/siTSP = 67; siGFP/ALP = 71; siGFP/siTSP/ALP = 71. ( i ) Effect of Wnt3 depletion on ectopic tentacle formation: Animals were electroporated with siRNAs specific for GFP or directed against Wnt3 and GFP followed by ALP- or control treatment as indicated. Ectopic tentacle formation was analyzed as described above ( h ). Number of polyps analyzed in each group was: siGFP: 74, siGFP/siWnt3: 76, siGFP/ALP: 79, and siGFP/siWnt3/ALP: 81. ( h and i show the results from three independent experiments. Each data point represents a single hydra, bars indicate the mean ± S.E.M. ***P value

Techniques Used: Functional Assay, In Situ Hybridization, Expressing, Transgenic Assay, Electroporation, ALP Assay

9) Product Images from "Superparamagnetic Iron Oxide Labeling of Stem Cells for MRI Tracking and Delivery in Cardiovascular Disease"

Article Title: Superparamagnetic Iron Oxide Labeling of Stem Cells for MRI Tracking and Delivery in Cardiovascular Disease

Journal:

doi: 10.1007/978-1-60761-705-1_11

BTX electroporation system ( a ) that can be used for magnetoelectroporation. This system may be operated by a switch ( b ) or foot pedal (not shown).
Figure Legend Snippet: BTX electroporation system ( a ) that can be used for magnetoelectroporation. This system may be operated by a switch ( b ) or foot pedal (not shown).

Techniques Used: Electroporation

10) Product Images from "14-3-3? Regulates Beclin 1 and Is Required for Autophagy"

Article Title: 14-3-3? Regulates Beclin 1 and Is Required for Autophagy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0010409

14-3-3τ is required for the expression of Beclin 1. (A) HEK293 and U2OS cells were transiently transfected with pSUPER-si14-3-3τ [15] or pSUPER expressing a scrambled sequence (siScr) [37] . Forty-four hr later, cells were harvested and RNA was extracted. Real-time RT-PCR analysis was performed using primers specific for Beclin 1, 14-3-3τ and GAPDH. The levels of Beclin 1 and 14-3-3τ were normalized to GAPDH levels and are expressed relative to the expression of the gene in the siScr control. The data shown represent the means ± standard deviations of triplicate samples. The p values are based on a paired two-tailed t test. (B) We established stable U2OS cell lines expressing an inducible siRNA against 14-3-3τ or a control GFP sequence under the control of tetracycline operon in pSUPERIOR.puro vector (OligoEngine). 14-3-3τ siRNA or a GFP siRNA was induced by doxycycline (DOX) (1 µg/ml), and cells were harvested for Western blot analysis using anti-Beclin 1, anti-14-3-3τ or anti-GAPDH antibody. (C) HEK293 cells were transiently transfected by three different pSUPER-14-3-3τ siRNA constructs (20 µg) by a calcium phosphate method. The cell lysates were harvested 48 hr later and analyzed by Western blot analysis. (D) HCT116 cells were transfected with 20 µg pSUPER-scrambled siRNA (siS) or si14-3-3τ (si14) along with a pcDNA3 empty vector or a vector expressing siRNA-resistant 14-3-3τ (res) by electroporation. The cell lysates were harvested 44 hr later and subject to Western blot analysis as indicated. (E) MCF7 cells were infected with a recombinant adenovirus expressing a control siGFP or si14-3-3τ (si14) at MOI of 100. Forty-two hr later, cells were harvested for Western blot analysis as indicated.
Figure Legend Snippet: 14-3-3τ is required for the expression of Beclin 1. (A) HEK293 and U2OS cells were transiently transfected with pSUPER-si14-3-3τ [15] or pSUPER expressing a scrambled sequence (siScr) [37] . Forty-four hr later, cells were harvested and RNA was extracted. Real-time RT-PCR analysis was performed using primers specific for Beclin 1, 14-3-3τ and GAPDH. The levels of Beclin 1 and 14-3-3τ were normalized to GAPDH levels and are expressed relative to the expression of the gene in the siScr control. The data shown represent the means ± standard deviations of triplicate samples. The p values are based on a paired two-tailed t test. (B) We established stable U2OS cell lines expressing an inducible siRNA against 14-3-3τ or a control GFP sequence under the control of tetracycline operon in pSUPERIOR.puro vector (OligoEngine). 14-3-3τ siRNA or a GFP siRNA was induced by doxycycline (DOX) (1 µg/ml), and cells were harvested for Western blot analysis using anti-Beclin 1, anti-14-3-3τ or anti-GAPDH antibody. (C) HEK293 cells were transiently transfected by three different pSUPER-14-3-3τ siRNA constructs (20 µg) by a calcium phosphate method. The cell lysates were harvested 48 hr later and analyzed by Western blot analysis. (D) HCT116 cells were transfected with 20 µg pSUPER-scrambled siRNA (siS) or si14-3-3τ (si14) along with a pcDNA3 empty vector or a vector expressing siRNA-resistant 14-3-3τ (res) by electroporation. The cell lysates were harvested 44 hr later and subject to Western blot analysis as indicated. (E) MCF7 cells were infected with a recombinant adenovirus expressing a control siGFP or si14-3-3τ (si14) at MOI of 100. Forty-two hr later, cells were harvested for Western blot analysis as indicated.

Techniques Used: Expressing, Transfection, Sequencing, Quantitative RT-PCR, Two Tailed Test, Plasmid Preparation, Western Blot, Construct, Electroporation, Infection, Recombinant

11) Product Images from "Potential Benefits of Sequential Inhibitor-Mutagen Treatments of RNA Virus Infections"

Article Title: Potential Benefits of Sequential Inhibitor-Mutagen Treatments of RNA Virus Infections

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000658

Effects of ribavirin (R) and guanidinium (GU) on the interference on FMDV replication exerted by capsid and 3D polymerase mutants. (A) Cells were mock-electroporated (control), or electroporated with 2 µg of pMT28 transcript (expresing FMDV C-S8c1), or co-electroporated with a mixture of transcripts obtained from pMT28 and from capsid mutant Q2027A and 3D polymerase mutant MD [63] . +R and +GU indicate the presence of R (1 mM) or GU (2 mM), respectively, as indicated in the box on the right. The ratio of pMT28 RNA to the total amount of mutant RNA was 1∶10, and the total amount of FMDV RNA was the same in each electroporation assay. Infectivity values were determined in triplicate at the indicated hours post-electroporation. Virus titers are expressed as percentage of the titers produced, relative to those produced by pMT28 RNA at the corresponding hours post-electroporation, taken as 100%. (B) Same as in (A) but using in the coelectroporations RNA from non-interfering polymerase mutants DMD and D3 [63] . In (A) and (B), no infectivity was obtained from the mock-electroporated samples (control box in dark grey, not visible). The origin of FMDV mutants, and procedures for titration of virus are detailed in Materials and Methods .
Figure Legend Snippet: Effects of ribavirin (R) and guanidinium (GU) on the interference on FMDV replication exerted by capsid and 3D polymerase mutants. (A) Cells were mock-electroporated (control), or electroporated with 2 µg of pMT28 transcript (expresing FMDV C-S8c1), or co-electroporated with a mixture of transcripts obtained from pMT28 and from capsid mutant Q2027A and 3D polymerase mutant MD [63] . +R and +GU indicate the presence of R (1 mM) or GU (2 mM), respectively, as indicated in the box on the right. The ratio of pMT28 RNA to the total amount of mutant RNA was 1∶10, and the total amount of FMDV RNA was the same in each electroporation assay. Infectivity values were determined in triplicate at the indicated hours post-electroporation. Virus titers are expressed as percentage of the titers produced, relative to those produced by pMT28 RNA at the corresponding hours post-electroporation, taken as 100%. (B) Same as in (A) but using in the coelectroporations RNA from non-interfering polymerase mutants DMD and D3 [63] . In (A) and (B), no infectivity was obtained from the mock-electroporated samples (control box in dark grey, not visible). The origin of FMDV mutants, and procedures for titration of virus are detailed in Materials and Methods .

Techniques Used: Mutagenesis, Electroporation, Infection, Produced, Titration

12) Product Images from "Efficacy of Antibacterial Peptides Against Peptide-Resistant MRSA Is Restored by Permeabilization of Bacteria Membranes"

Article Title: Efficacy of Antibacterial Peptides Against Peptide-Resistant MRSA Is Restored by Permeabilization of Bacteria Membranes

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.01745

Electroporation of MRSA with antimicrobial Peptides (AMPs). Electrocompetent WBG 8287 cells suspended in (A) 2.5 μg ml -1 melittin, (B) 50 μg ml -1 mel12-26, and (C) 2 μg ml -1 bac8c and electroporated immediately (EP). Melittin-resistant WBG 8287 suspended in (D) 10 μg ml -1 melittin or (E) 50 μg ml -1 mel12-26 and electroporated. Survival was measured as a percentage of zero peptide, unelectroporated controls. Data represent the mean of six samples and error bars represent standard deviation. ∗ P ≤ 0.05; ∗∗ P ≤ 0.001.
Figure Legend Snippet: Electroporation of MRSA with antimicrobial Peptides (AMPs). Electrocompetent WBG 8287 cells suspended in (A) 2.5 μg ml -1 melittin, (B) 50 μg ml -1 mel12-26, and (C) 2 μg ml -1 bac8c and electroporated immediately (EP). Melittin-resistant WBG 8287 suspended in (D) 10 μg ml -1 melittin or (E) 50 μg ml -1 mel12-26 and electroporated. Survival was measured as a percentage of zero peptide, unelectroporated controls. Data represent the mean of six samples and error bars represent standard deviation. ∗ P ≤ 0.05; ∗∗ P ≤ 0.001.

Techniques Used: Electroporation, Standard Deviation

13) Product Images from "Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation"

Article Title: Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-8-90

Electroporation of 3d mDC and 7d mDC with long MART-1/Melan-A peptide and MART-1/Melan-A-encoding ivt RNA . (A) 3d mDC and 7d mDC were electroporated (250 V, 150 μF) with 1 μg, 5 μg and 10 μg long MART-1/Melan-A peptide. After 24 h incubation at 37°C and 5% CO 2 , the DC were cocultured with A42 CTL for 24 h. (B) 2d iDC, 3d mDC and 7d mDC were electroporated (250 V, 150 μF) with 24 μg MART-1/Melan-A ivt RNA, incubated at 37°C for 24 h and cocultured with A42 CTL for 24 h (n = 3). (C) 3d mDC and 7d mDC were electroporated with 12 μg MART-1/Melan-A ivt RNA at different electroporation conditions (250 V, 150 μF and 300 V, 300 μF), respectively. 3 h after electroporation, mDC were stained intracellularly with a MART-1/Melan-A-specific antibody and analyzed by flow cytometry (n = 2). (D) 24 h after electroporation with MART-1/Melan-A ivt RNA, DC were cocultured with A42 CTL for 24 h (n = 2). The IFN-γ release of the A42 CTL was measured by IFN-γ-ELISA. The bars in A, B and D show mean values of triplicates with standard deviations (Rec.: recovery; n.d.: not detected).
Figure Legend Snippet: Electroporation of 3d mDC and 7d mDC with long MART-1/Melan-A peptide and MART-1/Melan-A-encoding ivt RNA . (A) 3d mDC and 7d mDC were electroporated (250 V, 150 μF) with 1 μg, 5 μg and 10 μg long MART-1/Melan-A peptide. After 24 h incubation at 37°C and 5% CO 2 , the DC were cocultured with A42 CTL for 24 h. (B) 2d iDC, 3d mDC and 7d mDC were electroporated (250 V, 150 μF) with 24 μg MART-1/Melan-A ivt RNA, incubated at 37°C for 24 h and cocultured with A42 CTL for 24 h (n = 3). (C) 3d mDC and 7d mDC were electroporated with 12 μg MART-1/Melan-A ivt RNA at different electroporation conditions (250 V, 150 μF and 300 V, 300 μF), respectively. 3 h after electroporation, mDC were stained intracellularly with a MART-1/Melan-A-specific antibody and analyzed by flow cytometry (n = 2). (D) 24 h after electroporation with MART-1/Melan-A ivt RNA, DC were cocultured with A42 CTL for 24 h (n = 2). The IFN-γ release of the A42 CTL was measured by IFN-γ-ELISA. The bars in A, B and D show mean values of triplicates with standard deviations (Rec.: recovery; n.d.: not detected).

Techniques Used: Electroporation, Incubation, CTL Assay, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

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Clone Assay:

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Centrifugation:

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Amplification:

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Mass Spectrometry:

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Stable Transfection:

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Acetylene Reduction Assay:

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Cytometry:

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Construct:

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Electrophoresis:

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Incubation:

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Article Title: Mutations in the 16S rRNA Genes of Helicobacter pylori Mediate Resistance to Tetracycline
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Article Title: Participation of fad and mbt Genes in Synthesis of Mycobactin in Mycobacterium smegmatis
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Expressing:

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Modification:

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Transformation Assay:

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Article Title: Targeting Tryptophan Decarboxylase to Selected Subcellular Compartments of Tobacco Plants Affects Enzyme Stability and in Vivo Function and Leads to a Lesion-Mimic Phenotype
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Article Title: Roles of the recJ and recN Genes in Homologous Recombination and DNA Repair Pathways of Neisseria gonorrhoeae
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Article Title: Naturally Occurring Lactococcal Plasmid pAH90 Links Bacteriophage Resistance and Mobility Functions to a Food-Grade Selectable Marker
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Derivative Assay:

Article Title: Introducing a Null Mutation in the Mouse K6? and K6? Genes Reveals Their Essential Structural Role in the Oral Mucosa
Article Snippet: The 5′ arm of homology is a 6.0-kb XbaI fragment derived from the upstream regulatory region of the cloned mouse K6α (mK6α) gene. .. DNA (20 μg) was transfected by electroporation into 2 × 107 exponentially growing 129/Sv CK35 embryonic stem (ES) cells ( ) using a BioRad Gene Pulser apparatus as described ( ).

Article Title: Properties of the Naturally Occurring Soluble Surface Glycoprotein of Ecotropic Murine Leukemia Virus: Binding Specificity and Possible Conformational Change after Binding to Receptor
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High Performance Liquid Chromatography:

Article Title: Hydra Mesoglea Proteome Identifies Thrombospondin as a Conserved Component Active in Head Organizer Restriction
Article Snippet: siRNAs (HPLC grade) specific for GFP and HmTSP (siGFP, scrambled siGFP, siTSP1 and siTSP2, see Supplementary Table for sequences) were purchased from Qiagen. .. For each reaction, 20 animals were transferred to electroporation cuvettes (4 mm gap, Bio-Rad) and excess liquid was removed.

Electroporation:

Article Title: Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)
Article Snippet: When targeting a gene within an operon, gene inactivation by plasmid insertion will result in the aberrant expression of genes located downstream of the mutation (polar effect) and should be taken into consideration when using this genetic approach. .. Genomic DNA from GAS strain to mutagenize Primers; design in step 1 High-fidelity DNA polymerase Reagents and equipment for PCR PCR purification kit (Promega) DNA of a temperature-sensitive plasmid conditionally replicative in GAS Restriction enzyme(s), DNA ligase and corresponding buffers Electrocompetent cells of E. coli strain for plasmid propagation (see Unit --) Electrocompetent cells of the GAS strain to mutagenize (see Basic Protocol 2) Gene Pulser Xcell Microbial System apparatus (Bio-Rad, Cat. No. 165-2662) Electroporation cuvettes (2 mm) (Bio-Rad, Cat. No. 165-2082) Todd-Hewitt Yeast (THY) broth for GAS growth (see recipe) Temperature-controlled CO2 incubator THY agar plates with appropriate antibiotic (see recipe) A set of primers targeting the gDNA surrounding the mutation .. 1 Design primers to amplify an internal fragment (within open reading frame) of the gene to mutagenize.

Article Title: Overexpression of Lactobacillus caseid-Hydroxyisocaproic Acid Dehydrogenase in Cheddar Cheese
Article Snippet: The cells were harvested by centrifugation at 5,000 × g , washed twice with sterile, distilled water, and suspended in 2.5 ml of ice-cold, sterile 30% polyethylene glycol 1450 (Sigma Chemical Co.). .. Three microliters of pHADH or pTRKH2 was mixed with 100 μl of cell suspension in a 0.2-cm electroporation cuvette and placed on ice for 3 min. An electric pulse was delivered in a Bio-Rad Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) set to the following parameters: 2.5 kV, 25 μF, and 200 Ω. .. After electroporation, 0.9 ml of warmed (37°C) MRS broth was added, and the cells were incubated at 37°C for 2 h. Transformants were collected on MRS agar that contained 5 μg of ERY per ml, and then cell lysates were prepared by the method of Anderson and McKay ( ) and uptake of pTRKH2 or pHADH was confirmed by agarose gel electrophoresis.

Article Title: Mutations in the 16S rRNA Genes of Helicobacter pylori Mediate Resistance to Tetracycline
Article Snippet: The method for electroporation of H . pylori was described previously ( ). .. DNA (approximately 0.5 μg) was mixed with 0.2 ml of cells, transferred to an electroporation cuvette (0.2-cm gap), and placed in a Bio-Rad Gene Pulser (Bio-Rad, Hercules, Calif.). .. A single pulse of 12.5 ms at 2.5 kV (12.5 kV/cm) with a 25-μF capacitor and a resistance of 600 Ω was used for transformation.

Article Title: Expression of MHC class II in T cells is associated with increased HIV-1 expression
Article Snippet: The pcDNACIITA-6, human MHC class II transactivator plasmid was prepared as described [ ]. .. Jurkat T cells (107 ) were transfected with 25 μg of pcDNACIITA-6 and 1 μg of neomycin DNA by electroporation using Gene Pulser Transfection Apparatus (BioRad, Richmond, CA) according to the manufacturer's protocol. .. Briefly, cells were transferred into an ice-cold Gene Pulser cuvette (0·4 cm) and subjected to a single pulse at 960 μF and 300 V. After shocking, the cuvette was incubated on ice for 10 min before cells were transferred to culture medium containing 10% FBS and 1 mg/ml G418 (Calbiochem, La Jolla, CA).

Article Title: Introducing a Null Mutation in the Mouse K6? and K6? Genes Reveals Their Essential Structural Role in the Oral Mucosa
Article Snippet: The targeting construct was linearized at the unique NotI site before transfection. .. DNA (20 μg) was transfected by electroporation into 2 × 107 exponentially growing 129/Sv CK35 embryonic stem (ES) cells ( ) using a BioRad Gene Pulser apparatus as described ( ). .. After electroporation, ES cells were screened for G418 (GIBCO BRL) and gancyclovir (Syntex Research) resistance for 10 d and genotyped.

Article Title: Gene Integration and Expression and Extracellular Secretion of Erwinia chrysanthemi Endoglucanase CelY (celY) and CelZ (celZ) in Ethanologenic Klebsiella oxytoca P2
Article Snippet: Constructions were confirmed by sequencing using the dideoxy method and a LI-COR Model 4000-L DNA sequencer with fluorescent primers. .. The E. chrysanthemi celY and celZ genes were introduced into K. oxytoca P2 by electroporation using a Bio-Rad Gene Pulser. .. Recombinants were selected on solid medium containing kanamycin (50 mg/liter) as previously described ( , ).

Article Title: Participation of fad and mbt Genes in Synthesis of Mycobactin in Mycobacterium smegmatis
Article Snippet: Introduction of plasmids into M. smegmatis was by electroporation ( ). .. Bacteria suspended in 10% glycerol electroporation buffer with 1 μg of DNA were subjected to a single pulse using the Bio-Rad Gene Pulser (Bio-Rad Laboratories) set at 2.5 kV, 25 μF, with the pulse controller resistance set at infinity. .. The content of the cuvette was diluted into 5 ml of TSB and incubated at 42°C.

Article Title: Characterization of an endogenous gene expressed in Aedes aegypti using an orally infectious recombinant Sindbis virus
Article Snippet: DNA was transcribed in vitro from the SP6 promoter, and RNA capping was achieved by adding a capping analog (Ambion, Inc.) to the mixture. .. This RNA was electroporated into 5 × 106 BHK-21 (baby hamster kidney) cells using a BioRad Gene Pulser set at 450 V, 125 µF, for 0.9 s. Cells and debris from the electroporation were immediately added to 4 ml of Leibovitz L-15 medium (Gibco BRL) supplemented with 10% fetal bovine serum in 25 cm2 tissue culture flasks. .. Viruses were harvested from BHK cells and titrated (plaque forming unit and tissue culture infectious dose 50% end-points (TCID50 )) 24 hours after transfection.

Article Title: Properties of the Naturally Occurring Soluble Surface Glycoprotein of Ecotropic Murine Leukemia Virus: Binding Specificity and Possible Conformational Change after Binding to Receptor
Article Snippet: FL21 cells are NIH 3T3 cells transfected with a DNA construct combining the Fv-4r MuLV region and its putative promoter region ( ). .. SIRC-NIH EcoR cells were established by transfection of SIRC cells with pcDNA-NIH EcoR (see below) by the electroporation method (Gene Pulser; Bio-Rad), and transformants were selected in a medium containing 1 mg of Geneticin (Sigma) per ml. .. A few SIRC-NIH EcoR transformant cell lines analyzed in this study were independently derived from single primary colonies.

Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae
Article Snippet: All gene targeting, gene cloning and plasmid propagation procedures were performed in E. coli DH10B [Invitrogen, genotype: F- mcr A Δ(mrr -hsd RMS-mcr BC) Φ80lac ZΔM15 Δlac X74 rec A1 end A1 ara D139 Δ(ara leu ) 7697 gal U gal K rps L nup G λ-], incubating plates and liquid cultures at 37°C. .. All oligonucleotides, linear dsDNA and plasmid DNA were transformed into E. coli cells by electroporation using a MicroPulser electroporator with 1 mm gap electroporation cuvettes (BioRad). .. Transformed cells were plated onto Luria-Bertani (LB) agar (USB), and liquid cultures were grown in LB medium (USB); supplementing with kanamycin (Kan, 50 μg/ml, USB), ampicillin (Amp, 50 μg/ml, USB) and/or chloramphenicol (Cm, 30 μg/ml, Sigma) for plasmid maintenance, where appropriate.

Article Title: Targeting Tryptophan Decarboxylase to Selected Subcellular Compartments of Tobacco Plants Affects Enzyme Stability and in Vivo Function and Leads to a Lesion-Mimic Phenotype
Article Snippet: The recombinant tdc cDNAs including the 5′ targeting sequences and the 3′ tags were subcloned as a Eco RI/XbaI fragment into the pSS plant expression vector ( ) between the constitutive CaMV 35S double enhanced promoter and the CaMV terminator sequence, resulting in the plant expression vectors pT-CHL, pT-CYT, and pT-ER for targeting the TDC protein to the chloroplast, cytosol, and ER of plant cells, respectively (Fig. ). .. The plant expression vectors were introduced into A. tumefaciens GV3101 cells by electroporation using a Gene Pulser II electroporation system (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. .. In preliminary experiments, TDC expression and in vivo function was evaluated using a transient expression assay of vacuum-infiltrated tobacco leaves with recombinant Agrobacteria ( ).

Article Title: Roles of the recJ and recN Genes in Homologous Recombination and DNA Repair Pathways of Neisseria gonorrhoeae
Article Snippet: Enzymes were used as specified by the manufacturers (Promega Corp. and New England Biolabs). .. E. coli strains were transformed by electroporation using the Gene Pulser II electroporation system (Bio-Rad Laboratories) as specified by the manufacturer. .. For Southern blot analysis, DNA was transferred to Magnagraph nylon membrane as specified by the manufacturer (Micro Separations Inc.).

Article Title: Construction of a Fully Retargeted Herpes Simplex Virus 1 Recombinant Capable of Entering Cells Solely via Human Epidermal Growth Factor Receptor 2
Article Snippet: The procedure applied to generate recombinant genomes in E. coli was essentially as described previously, with slight modifications ( , , ). .. Briefly, electrocompetent E. coli DH10B harboring the relevant gD-minus HSV-BAC genomes was electroporated with the shuttle vector in 0.2-cm electroporation cuvettes (Bio-Rad) at 200 Ω, 25 μF, and 2.5 kV; plated on LB agar containing 25 μg/ml Kan (the shuttle vector's marker) and 20 μg/ml Cam (the BAC's marker); and incubated at 30°C overnight to allow the expression of RecA from the shuttle vector. .. The clones were replated onto LB plus Kan plus Cam at 43°C to allow the identification of those harboring the cointegrates (visible as large colonies compared to the temperature-sensitive “small-colony” phenotype determined by nonintegrated shuttle vectors).

Article Title: Hydra Mesoglea Proteome Identifies Thrombospondin as a Conserved Component Active in Head Organizer Restriction
Article Snippet: In brief, animals from a daily-fed culture of chimeric ecto-GFP/endo-RFP transgenic animals were washed twice with ultrapure water. .. For each reaction, 20 animals were transferred to electroporation cuvettes (4 mm gap, Bio-Rad) and excess liquid was removed. .. After adding 200 µl of sterile ultrapure water containing either 3 µM of siGFP (1 µM siGFP and 2 µM scrambled siGFP) or a combination of siGFP, siTSP1, and siTSP2 (1 µM each) animals were allowed to relax for 20 min at room temperature.

Article Title: Approaches for functional analysis of flagellar proteins in African trypanosomes
Article Snippet: BioRad® Gene Pulser II gene pulser. .. BioRad® electroporation cuvettes 0.4 cm. .. Electroporation Medium (“EM”) Reference: [ ]

Article Title: Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes
Article Snippet: T4 DNA ligase (Invitrogen or NEB). .. Electroporation cuvettes (Bio-Rad). .. Competent DH5α cells (Invitrogen).

Article Title: Deletion of murine kininogen gene 1 (mKng1) causes loss of plasma kininogen and delays thrombosis
Article Snippet: Paragraph title: Electroporation and selection of mKng1−/− ES cells ... Briefly, cuvettes containing 0.8 mL cells at a concentration of 7 × 106 cells/mL and 40 μg of linearized DNA were pulsed at 0.240 kV and 500 μF using a Bio-Rad gene pulser (Bio-Rad, Cupertino, CA).

Article Title: Naturally Occurring Lactococcal Plasmid pAH90 Links Bacteriophage Resistance and Mobility Functions to a Food-Grade Selectable Marker
Article Snippet: Paragraph title: Electroporation and transformation. ... L. lactis MG1614 and DPC3333 were made competent and electrotransformed using a Bio-Rad gene pulser apparatus (Bio-Rad Corp., Richmond, Calif.) as described previously ( ).

Article Title: Biocontrol of the Sugarcane Borer Eldana saccharina by Expression of the Bacillus thuringiensis cry1Ac7 and Serratia marcescens chiA Genes in Sugarcane-Associated Bacteria
Article Snippet: Paragraph title: Bacterial transformation by electroporation and conjugation. ... Cells harvested at mid-exponential phase and washed three times in 300 mM sucrose were electroporated in 40-μl volumes with 1 to 3 μg of DNA using a Bio-Rad Gene Pulser and controller set at 25 μF, 2.5 kV, and 200 Ω. Phenotypic expression was carried out at 30°C for 2 h in 1 ml of LB or JNFb medium (for P. fluorescens and H. seropedicae , respectively).

Article Title: A Novel Testicular RhoGAP-Domain Protein Induces Apoptosis
Article Snippet: Briefly, the complete tGAP1 coding region (sense orientation; 3879 base-pair [bp] Kpn I/ Not I restriction fragment), partial tGAP1 antisense orientation (3166-bp Xho I/ Sal I restriction fragment), and human p53 (1191-bp Xba I/ Bgl II restriction fragment) were subcloned into the pAdTrack-CMV shuttle vector. .. The shuttle-vector plasmids and a shuttle vector without insert were linearized with Pme I and transformed into Escherichia coli strain BJ5183 by electroporation in a Bio-Rad Gene Pulser electroporator (Bio-Rad, Mississauga, ON, Canada): BJ5183 stably contains the adenoviral backbone vector pAdEasy-1. .. Bacterial colonies containing recombinants were selected with kanamycin (50 μg/ml) and screened by restriction endonuclease digestions ( Pac I, Eco RI, and Bam HI).

Transfection:

Article Title: Expression of MHC class II in T cells is associated with increased HIV-1 expression
Article Snippet: The pcDNACIITA-6, human MHC class II transactivator plasmid was prepared as described [ ]. .. Jurkat T cells (107 ) were transfected with 25 μg of pcDNACIITA-6 and 1 μg of neomycin DNA by electroporation using Gene Pulser Transfection Apparatus (BioRad, Richmond, CA) according to the manufacturer's protocol. .. Briefly, cells were transferred into an ice-cold Gene Pulser cuvette (0·4 cm) and subjected to a single pulse at 960 μF and 300 V. After shocking, the cuvette was incubated on ice for 10 min before cells were transferred to culture medium containing 10% FBS and 1 mg/ml G418 (Calbiochem, La Jolla, CA).

Article Title: Introducing a Null Mutation in the Mouse K6? and K6? Genes Reveals Their Essential Structural Role in the Oral Mucosa
Article Snippet: The targeting construct was linearized at the unique NotI site before transfection. .. DNA (20 μg) was transfected by electroporation into 2 × 107 exponentially growing 129/Sv CK35 embryonic stem (ES) cells ( ) using a BioRad Gene Pulser apparatus as described ( ). .. After electroporation, ES cells were screened for G418 (GIBCO BRL) and gancyclovir (Syntex Research) resistance for 10 d and genotyped.

Article Title: Properties of the Naturally Occurring Soluble Surface Glycoprotein of Ecotropic Murine Leukemia Virus: Binding Specificity and Possible Conformational Change after Binding to Receptor
Article Snippet: FL21 cells are NIH 3T3 cells transfected with a DNA construct combining the Fv-4r MuLV region and its putative promoter region ( ). .. SIRC-NIH EcoR cells were established by transfection of SIRC cells with pcDNA-NIH EcoR (see below) by the electroporation method (Gene Pulser; Bio-Rad), and transformants were selected in a medium containing 1 mg of Geneticin (Sigma) per ml. .. A few SIRC-NIH EcoR transformant cell lines analyzed in this study were independently derived from single primary colonies.

Article Title: Approaches for functional analysis of flagellar proteins in African trypanosomes
Article Snippet: Paragraph title: IV.B Transfection reagents ... BioRad® electroporation cuvettes 0.4 cm.

Article Title: A Novel Testicular RhoGAP-Domain Protein Induces Apoptosis
Article Snippet: The shuttle-vector plasmids and a shuttle vector without insert were linearized with Pme I and transformed into Escherichia coli strain BJ5183 by electroporation in a Bio-Rad Gene Pulser electroporator (Bio-Rad, Mississauga, ON, Canada): BJ5183 stably contains the adenoviral backbone vector pAdEasy-1. .. The recombinant adenoviral constructs were transformed into XL1Blue bacteria for large-scale amplification.

Sequencing:

Article Title: Overexpression of Lactobacillus caseid-Hydroxyisocaproic Acid Dehydrogenase in Cheddar Cheese
Article Snippet: Transformants were selected on Luria-Bertani agar that contained 500 μg of ERY per ml, plasmid DNA was isolated from Eryr CFU by the alkaline lysis method , and the presence of dhic insert DNA was confirmed by agarose gel electrophoresis and DNA sequence analysis. .. Three microliters of pHADH or pTRKH2 was mixed with 100 μl of cell suspension in a 0.2-cm electroporation cuvette and placed on ice for 3 min. An electric pulse was delivered in a Bio-Rad Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) set to the following parameters: 2.5 kV, 25 μF, and 200 Ω.

Article Title: Gene Integration and Expression and Extracellular Secretion of Erwinia chrysanthemi Endoglucanase CelY (celY) and CelZ (celZ) in Ethanologenic Klebsiella oxytoca P2
Article Snippet: Constructions were confirmed by sequencing using the dideoxy method and a LI-COR Model 4000-L DNA sequencer with fluorescent primers. .. The E. chrysanthemi celY and celZ genes were introduced into K. oxytoca P2 by electroporation using a Bio-Rad Gene Pulser.

Southern Blot:

Article Title: Introducing a Null Mutation in the Mouse K6? and K6? Genes Reveals Their Essential Structural Role in the Oral Mucosa
Article Snippet: DNA (20 μg) was transfected by electroporation into 2 × 107 exponentially growing 129/Sv CK35 embryonic stem (ES) cells ( ) using a BioRad Gene Pulser apparatus as described ( ). .. Chimeric males, identified by their agouti coat color, were mated with (C57Bl/6xDBA2)F1 females, and the resulting mice heterozygous for the disrupted K6 allele were interbred to obtain the K6 deficient mice.

Article Title: Deletion of murine kininogen gene 1 (mKng1) causes loss of plasma kininogen and delays thrombosis
Article Snippet: Briefly, cuvettes containing 0.8 mL cells at a concentration of 7 × 106 cells/mL and 40 μg of linearized DNA were pulsed at 0.240 kV and 500 μF using a Bio-Rad gene pulser (Bio-Rad, Cupertino, CA). .. Briefly, cuvettes containing 0.8 mL cells at a concentration of 7 × 106 cells/mL and 40 μg of linearized DNA were pulsed at 0.240 kV and 500 μF using a Bio-Rad gene pulser (Bio-Rad, Cupertino, CA).

Infection:

Article Title: Properties of the Naturally Occurring Soluble Surface Glycoprotein of Ecotropic Murine Leukemia Virus: Binding Specificity and Possible Conformational Change after Binding to Receptor
Article Snippet: SIRC-NIH EcoR cells were established by transfection of SIRC cells with pcDNA-NIH EcoR (see below) by the electroporation method (Gene Pulser; Bio-Rad), and transformants were selected in a medium containing 1 mg of Geneticin (Sigma) per ml. .. NIH 3T3-HmFCR cells were NIH 3T3 cells transfected with a plasmid, pHmFCR , with Lipofectamine reagent (Gibco BRL) and were selected with 100 μg of hygromycin-B (Wako Pure Chemical Industries, Ltd.) per ml.

Polymerase Chain Reaction:

Article Title: Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)
Article Snippet: When targeting a gene within an operon, gene inactivation by plasmid insertion will result in the aberrant expression of genes located downstream of the mutation (polar effect) and should be taken into consideration when using this genetic approach. .. Genomic DNA from GAS strain to mutagenize Primers; design in step 1 High-fidelity DNA polymerase Reagents and equipment for PCR PCR purification kit (Promega) DNA of a temperature-sensitive plasmid conditionally replicative in GAS Restriction enzyme(s), DNA ligase and corresponding buffers Electrocompetent cells of E. coli strain for plasmid propagation (see Unit --) Electrocompetent cells of the GAS strain to mutagenize (see Basic Protocol 2) Gene Pulser Xcell Microbial System apparatus (Bio-Rad, Cat. No. 165-2662) Electroporation cuvettes (2 mm) (Bio-Rad, Cat. No. 165-2082) Todd-Hewitt Yeast (THY) broth for GAS growth (see recipe) Temperature-controlled CO2 incubator THY agar plates with appropriate antibiotic (see recipe) A set of primers targeting the gDNA surrounding the mutation .. 1 Design primers to amplify an internal fragment (within open reading frame) of the gene to mutagenize.

Article Title: Mutations in the 16S rRNA Genes of Helicobacter pylori Mediate Resistance to Tetracycline
Article Snippet: The DNA samples used in the natural transformation experiments were purified PCR products. .. DNA (approximately 0.5 μg) was mixed with 0.2 ml of cells, transferred to an electroporation cuvette (0.2-cm gap), and placed in a Bio-Rad Gene Pulser (Bio-Rad, Hercules, Calif.).

Article Title: Gene Integration and Expression and Extracellular Secretion of Erwinia chrysanthemi Endoglucanase CelY (celY) and CelZ (celZ) in Ethanologenic Klebsiella oxytoca P2
Article Snippet: The ribosome-binding site and promoterless coding region of celY were amplified by the PCR using pMH18 as the template with the following primer pair: N terminus, 5′-CTGTTCCGTTACCAACAC-3′, and C terminus, 5′-GTGAATGGGATCACGAGT-3′. .. The E. chrysanthemi celY and celZ genes were introduced into K. oxytoca P2 by electroporation using a Bio-Rad Gene Pulser.

Article Title: Characterization of an endogenous gene expressed in Aedes aegypti using an orally infectious recombinant Sindbis virus
Article Snippet: This RNA was electroporated into 5 × 106 BHK-21 (baby hamster kidney) cells using a BioRad Gene Pulser set at 450 V, 125 µF, for 0.9 s. Cells and debris from the electroporation were immediately added to 4 ml of Leibovitz L-15 medium (Gibco BRL) supplemented with 10% fetal bovine serum in 25 cm2 tissue culture flasks. .. Approximately 5–8 × 107 plaque forming units or 7.2–8 log10 TCID50 per ml of MRE/2'3J, MRE/2'3J/ppA, and MRE/2'3J/ppC viruses were obtained.

Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae
Article Snippet: Plasmid pJB1 was constructed by Dr. John Beaber and was generously supplied by Prof. Matthew Waldor (Harvard Medical School and HHMI). pJB1 contains the region of the SXT integrating conjugative element (ICE) that includes the ssb (s064) , bet (s065) and exo (s066) genes, was used as the template for the PCR-amplification of all SXT genes. .. All oligonucleotides, linear dsDNA and plasmid DNA were transformed into E. coli cells by electroporation using a MicroPulser electroporator with 1 mm gap electroporation cuvettes (BioRad).

Article Title: Roles of the recJ and recN Genes in Homologous Recombination and DNA Repair Pathways of Neisseria gonorrhoeae
Article Snippet: E. coli strains were transformed by electroporation using the Gene Pulser II electroporation system (Bio-Rad Laboratories) as specified by the manufacturer. .. E. coli strains were transformed by electroporation using the Gene Pulser II electroporation system (Bio-Rad Laboratories) as specified by the manufacturer.

Article Title: Construction of a Fully Retargeted Herpes Simplex Virus 1 Recombinant Capable of Entering Cells Solely via Human Epidermal Growth Factor Receptor 2
Article Snippet: Briefly, electrocompetent E. coli DH10B harboring the relevant gD-minus HSV-BAC genomes was electroporated with the shuttle vector in 0.2-cm electroporation cuvettes (Bio-Rad) at 200 Ω, 25 μF, and 2.5 kV; plated on LB agar containing 25 μg/ml Kan (the shuttle vector's marker) and 20 μg/ml Cam (the BAC's marker); and incubated at 30°C overnight to allow the expression of RecA from the shuttle vector. .. Subsequently, the cointegrates were allowed to resolve by plating the clones onto LB plus Cam at 30°C, and clones containing the resolved HSV-BAC were selected on LB-plus-Cam plates supplemented with 10% sucrose.

Article Title: Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes
Article Snippet: Paragraph title: 2.2. Cloning of PCR Fragments into a pUC18 Plasmid ... Electroporation cuvettes (Bio-Rad).

Article Title: Deletion of murine kininogen gene 1 (mKng1) causes loss of plasma kininogen and delays thrombosis
Article Snippet: Briefly, cuvettes containing 0.8 mL cells at a concentration of 7 × 106 cells/mL and 40 μg of linearized DNA were pulsed at 0.240 kV and 500 μF using a Bio-Rad gene pulser (Bio-Rad, Cupertino, CA). .. Briefly, cuvettes containing 0.8 mL cells at a concentration of 7 × 106 cells/mL and 40 μg of linearized DNA were pulsed at 0.240 kV and 500 μF using a Bio-Rad gene pulser (Bio-Rad, Cupertino, CA).

Electroporation Bacterial Transformation:

Article Title: Biocontrol of the Sugarcane Borer Eldana saccharina by Expression of the Bacillus thuringiensis cry1Ac7 and Serratia marcescens chiA Genes in Sugarcane-Associated Bacteria
Article Snippet: Paragraph title: Bacterial transformation by electroporation and conjugation. ... Cells harvested at mid-exponential phase and washed three times in 300 mM sucrose were electroporated in 40-μl volumes with 1 to 3 μg of DNA using a Bio-Rad Gene Pulser and controller set at 25 μF, 2.5 kV, and 200 Ω. Phenotypic expression was carried out at 30°C for 2 h in 1 ml of LB or JNFb medium (for P. fluorescens and H. seropedicae , respectively).

Injection:

Article Title: Introducing a Null Mutation in the Mouse K6? and K6? Genes Reveals Their Essential Structural Role in the Oral Mucosa
Article Snippet: DNA (20 μg) was transfected by electroporation into 2 × 107 exponentially growing 129/Sv CK35 embryonic stem (ES) cells ( ) using a BioRad Gene Pulser apparatus as described ( ). .. After electroporation, ES cells were screened for G418 (GIBCO BRL) and gancyclovir (Syntex Research) resistance for 10 d and genotyped.

Binding Assay:

Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae
Article Snippet: All arabinose-inducible plasmids constructed here are derivatives of pBAD-ETγ (see Additional File ), and contain identical 4503 bp NcoI/HindIII backbone fragments, which houses the ColE1 ori , bla ampicillin resistance gene, araC repressor, PBAD operator/promoter region, and ribosome binding site immediately upstream of the adjacent NcoI and NdeI restriction sites. .. All oligonucleotides, linear dsDNA and plasmid DNA were transformed into E. coli cells by electroporation using a MicroPulser electroporator with 1 mm gap electroporation cuvettes (BioRad).

Conjugation Assay:

Article Title: Gene Integration and Expression and Extracellular Secretion of Erwinia chrysanthemi Endoglucanase CelY (celY) and CelZ (celZ) in Ethanologenic Klebsiella oxytoca P2
Article Snippet: The E. chrysanthemi out genes (pCPP2006) were transferred by conjugation using pRK2013 for mobilization ( ). .. The E. chrysanthemi celY and celZ genes were introduced into K. oxytoca P2 by electroporation using a Bio-Rad Gene Pulser.

Article Title: Biocontrol of the Sugarcane Borer Eldana saccharina by Expression of the Bacillus thuringiensis cry1Ac7 and Serratia marcescens chiA Genes in Sugarcane-Associated Bacteria
Article Snippet: Paragraph title: Bacterial transformation by electroporation and conjugation. ... Cells harvested at mid-exponential phase and washed three times in 300 mM sucrose were electroporated in 40-μl volumes with 1 to 3 μg of DNA using a Bio-Rad Gene Pulser and controller set at 25 μF, 2.5 kV, and 200 Ω. Phenotypic expression was carried out at 30°C for 2 h in 1 ml of LB or JNFb medium (for P. fluorescens and H. seropedicae , respectively).

Mutagenesis:

Article Title: Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)
Article Snippet: When targeting a gene within an operon, gene inactivation by plasmid insertion will result in the aberrant expression of genes located downstream of the mutation (polar effect) and should be taken into consideration when using this genetic approach. .. Genomic DNA from GAS strain to mutagenize Primers; design in step 1 High-fidelity DNA polymerase Reagents and equipment for PCR PCR purification kit (Promega) DNA of a temperature-sensitive plasmid conditionally replicative in GAS Restriction enzyme(s), DNA ligase and corresponding buffers Electrocompetent cells of E. coli strain for plasmid propagation (see Unit --) Electrocompetent cells of the GAS strain to mutagenize (see Basic Protocol 2) Gene Pulser Xcell Microbial System apparatus (Bio-Rad, Cat. No. 165-2662) Electroporation cuvettes (2 mm) (Bio-Rad, Cat. No. 165-2082) Todd-Hewitt Yeast (THY) broth for GAS growth (see recipe) Temperature-controlled CO2 incubator THY agar plates with appropriate antibiotic (see recipe) A set of primers targeting the gDNA surrounding the mutation .. 1 Design primers to amplify an internal fragment (within open reading frame) of the gene to mutagenize.

Isolation:

Article Title: Overexpression of Lactobacillus caseid-Hydroxyisocaproic Acid Dehydrogenase in Cheddar Cheese
Article Snippet: Transformants were selected on Luria-Bertani agar that contained 500 μg of ERY per ml, plasmid DNA was isolated from Eryr CFU by the alkaline lysis method , and the presence of dhic insert DNA was confirmed by agarose gel electrophoresis and DNA sequence analysis. .. Three microliters of pHADH or pTRKH2 was mixed with 100 μl of cell suspension in a 0.2-cm electroporation cuvette and placed on ice for 3 min. An electric pulse was delivered in a Bio-Rad Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) set to the following parameters: 2.5 kV, 25 μF, and 200 Ω.

Article Title: Participation of fad and mbt Genes in Synthesis of Mycobactin in Mycobacterium smegmatis
Article Snippet: Isolation of plasmid DNA from M. smegmatis ( ) was essentially like that from E. coli , i.e., alkaline lysis, except that the cells were first disrupted (beaten for 2 min) by glass beads in a Mini-BeadBeater (Biospec Products, Bartlesville, Okla.). .. Bacteria suspended in 10% glycerol electroporation buffer with 1 μg of DNA were subjected to a single pulse using the Bio-Rad Gene Pulser (Bio-Rad Laboratories) set at 2.5 kV, 25 μF, with the pulse controller resistance set at infinity.

Article Title: Characterization of an endogenous gene expressed in Aedes aegypti using an orally infectious recombinant Sindbis virus
Article Snippet: DNA was isolated using the QIAfilter Midi Kit (Qiagen) according to the manufacturer's instructions. .. This RNA was electroporated into 5 × 106 BHK-21 (baby hamster kidney) cells using a BioRad Gene Pulser set at 450 V, 125 µF, for 0.9 s. Cells and debris from the electroporation were immediately added to 4 ml of Leibovitz L-15 medium (Gibco BRL) supplemented with 10% fetal bovine serum in 25 cm2 tissue culture flasks.

Flow Cytometry:

Article Title: Expression of MHC class II in T cells is associated with increased HIV-1 expression
Article Snippet: Jurkat T cells (107 ) were transfected with 25 μg of pcDNACIITA-6 and 1 μg of neomycin DNA by electroporation using Gene Pulser Transfection Apparatus (BioRad, Richmond, CA) according to the manufacturer's protocol. .. Jurkat T cells (107 ) were transfected with 25 μg of pcDNACIITA-6 and 1 μg of neomycin DNA by electroporation using Gene Pulser Transfection Apparatus (BioRad, Richmond, CA) according to the manufacturer's protocol.

Purification:

Article Title: Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)
Article Snippet: When targeting a gene within an operon, gene inactivation by plasmid insertion will result in the aberrant expression of genes located downstream of the mutation (polar effect) and should be taken into consideration when using this genetic approach. .. Genomic DNA from GAS strain to mutagenize Primers; design in step 1 High-fidelity DNA polymerase Reagents and equipment for PCR PCR purification kit (Promega) DNA of a temperature-sensitive plasmid conditionally replicative in GAS Restriction enzyme(s), DNA ligase and corresponding buffers Electrocompetent cells of E. coli strain for plasmid propagation (see Unit --) Electrocompetent cells of the GAS strain to mutagenize (see Basic Protocol 2) Gene Pulser Xcell Microbial System apparatus (Bio-Rad, Cat. No. 165-2662) Electroporation cuvettes (2 mm) (Bio-Rad, Cat. No. 165-2082) Todd-Hewitt Yeast (THY) broth for GAS growth (see recipe) Temperature-controlled CO2 incubator THY agar plates with appropriate antibiotic (see recipe) A set of primers targeting the gDNA surrounding the mutation .. 1 Design primers to amplify an internal fragment (within open reading frame) of the gene to mutagenize.

Article Title: Mutations in the 16S rRNA Genes of Helicobacter pylori Mediate Resistance to Tetracycline
Article Snippet: The DNA samples used in the natural transformation experiments were purified PCR products. .. DNA (approximately 0.5 μg) was mixed with 0.2 ml of cells, transferred to an electroporation cuvette (0.2-cm gap), and placed in a Bio-Rad Gene Pulser (Bio-Rad, Hercules, Calif.).

Article Title: Participation of fad and mbt Genes in Synthesis of Mycobactin in Mycobacterium smegmatis
Article Snippet: Plasmid DNA from E. coli cells was prepared by the alkaline lysis method ( ) and was purified by RNase treatment and phenol extraction. .. Bacteria suspended in 10% glycerol electroporation buffer with 1 μg of DNA were subjected to a single pulse using the Bio-Rad Gene Pulser (Bio-Rad Laboratories) set at 2.5 kV, 25 μF, with the pulse controller resistance set at infinity.

Article Title: Characterization of an endogenous gene expressed in Aedes aegypti using an orally infectious recombinant Sindbis virus
Article Snippet: This RNA was electroporated into 5 × 106 BHK-21 (baby hamster kidney) cells using a BioRad Gene Pulser set at 450 V, 125 µF, for 0.9 s. Cells and debris from the electroporation were immediately added to 4 ml of Leibovitz L-15 medium (Gibco BRL) supplemented with 10% fetal bovine serum in 25 cm2 tissue culture flasks. .. Approximately 5–8 × 107 plaque forming units or 7.2–8 log10 TCID50 per ml of MRE/2'3J, MRE/2'3J/ppA, and MRE/2'3J/ppC viruses were obtained.

Article Title: Roles of the recJ and recN Genes in Homologous Recombination and DNA Repair Pathways of Neisseria gonorrhoeae
Article Snippet: E. coli strains were transformed by electroporation using the Gene Pulser II electroporation system (Bio-Rad Laboratories) as specified by the manufacturer. .. DNA sequence analysis was performed using Lasergene software (DNASTAR, Inc.) and VectorNTI software (Informax, Inc.).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Characterization of an endogenous gene expressed in Aedes aegypti using an orally infectious recombinant Sindbis virus
Article Snippet: This RNA was electroporated into 5 × 106 BHK-21 (baby hamster kidney) cells using a BioRad Gene Pulser set at 450 V, 125 µF, for 0.9 s. Cells and debris from the electroporation were immediately added to 4 ml of Leibovitz L-15 medium (Gibco BRL) supplemented with 10% fetal bovine serum in 25 cm2 tissue culture flasks. .. Approximately 5–8 × 107 plaque forming units or 7.2–8 log10 TCID50 per ml of MRE/2'3J, MRE/2'3J/ppA, and MRE/2'3J/ppC viruses were obtained.

Selection:

Article Title: Introducing a Null Mutation in the Mouse K6? and K6? Genes Reveals Their Essential Structural Role in the Oral Mucosa
Article Snippet: Plasmid NeoTKXho, with functional PGK-Neo (Neo) and MC1TK (TK [thymidine kinase]) cassettes for selection, was obtained from Dr. Jeremy Nathans (Johns Hopkins University, Baltimore, MD) and used to generate the targeting vector. .. DNA (20 μg) was transfected by electroporation into 2 × 107 exponentially growing 129/Sv CK35 embryonic stem (ES) cells ( ) using a BioRad Gene Pulser apparatus as described ( ).

Article Title: Deletion of murine kininogen gene 1 (mKng1) causes loss of plasma kininogen and delays thrombosis
Article Snippet: Paragraph title: Electroporation and selection of mKng1−/− ES cells ... Briefly, cuvettes containing 0.8 mL cells at a concentration of 7 × 106 cells/mL and 40 μg of linearized DNA were pulsed at 0.240 kV and 500 μF using a Bio-Rad gene pulser (Bio-Rad, Cupertino, CA).

Mouse Assay:

Article Title: Introducing a Null Mutation in the Mouse K6? and K6? Genes Reveals Their Essential Structural Role in the Oral Mucosa
Article Snippet: Paragraph title: Construction of Targeting Vector and Generation of Transgenic Mice ... DNA (20 μg) was transfected by electroporation into 2 × 107 exponentially growing 129/Sv CK35 embryonic stem (ES) cells ( ) using a BioRad Gene Pulser apparatus as described ( ).

Article Title: Properties of the Naturally Occurring Soluble Surface Glycoprotein of Ecotropic Murine Leukemia Virus: Binding Specificity and Possible Conformational Change after Binding to Receptor
Article Snippet: Paragraph title: Mice and cells. ... SIRC-NIH EcoR cells were established by transfection of SIRC cells with pcDNA-NIH EcoR (see below) by the electroporation method (Gene Pulser; Bio-Rad), and transformants were selected in a medium containing 1 mg of Geneticin (Sigma) per ml.

Plasmid Preparation:

Article Title: Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)
Article Snippet: When targeting a gene within an operon, gene inactivation by plasmid insertion will result in the aberrant expression of genes located downstream of the mutation (polar effect) and should be taken into consideration when using this genetic approach. .. Genomic DNA from GAS strain to mutagenize Primers; design in step 1 High-fidelity DNA polymerase Reagents and equipment for PCR PCR purification kit (Promega) DNA of a temperature-sensitive plasmid conditionally replicative in GAS Restriction enzyme(s), DNA ligase and corresponding buffers Electrocompetent cells of E. coli strain for plasmid propagation (see Unit --) Electrocompetent cells of the GAS strain to mutagenize (see Basic Protocol 2) Gene Pulser Xcell Microbial System apparatus (Bio-Rad, Cat. No. 165-2662) Electroporation cuvettes (2 mm) (Bio-Rad, Cat. No. 165-2082) Todd-Hewitt Yeast (THY) broth for GAS growth (see recipe) Temperature-controlled CO2 incubator THY agar plates with appropriate antibiotic (see recipe) A set of primers targeting the gDNA surrounding the mutation .. 1 Design primers to amplify an internal fragment (within open reading frame) of the gene to mutagenize.

Article Title: Overexpression of Lactobacillus caseid-Hydroxyisocaproic Acid Dehydrogenase in Cheddar Cheese
Article Snippet: The pTRKH2: dhic plasmid construct from a representative clone was selected for further work and designated pHADH. .. Three microliters of pHADH or pTRKH2 was mixed with 100 μl of cell suspension in a 0.2-cm electroporation cuvette and placed on ice for 3 min. An electric pulse was delivered in a Bio-Rad Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) set to the following parameters: 2.5 kV, 25 μF, and 200 Ω.

Article Title: Introducing a Null Mutation in the Mouse K6? and K6? Genes Reveals Their Essential Structural Role in the Oral Mucosa
Article Snippet: Paragraph title: Construction of Targeting Vector and Generation of Transgenic Mice ... DNA (20 μg) was transfected by electroporation into 2 × 107 exponentially growing 129/Sv CK35 embryonic stem (ES) cells ( ) using a BioRad Gene Pulser apparatus as described ( ).

Article Title: Gene Integration and Expression and Extracellular Secretion of Erwinia chrysanthemi Endoglucanase CelY (celY) and CelZ (celZ) in Ethanologenic Klebsiella oxytoca P2
Article Snippet: The E. chrysanthemi celY and celZ genes were introduced into K. oxytoca P2 by electroporation using a Bio-Rad Gene Pulser. .. The E. chrysanthemi celY and celZ genes were introduced into K. oxytoca P2 by electroporation using a Bio-Rad Gene Pulser.

Article Title: Participation of fad and mbt Genes in Synthesis of Mycobactin in Mycobacterium smegmatis
Article Snippet: Isolation of plasmid DNA from M. smegmatis ( ) was essentially like that from E. coli , i.e., alkaline lysis, except that the cells were first disrupted (beaten for 2 min) by glass beads in a Mini-BeadBeater (Biospec Products, Bartlesville, Okla.). .. Bacteria suspended in 10% glycerol electroporation buffer with 1 μg of DNA were subjected to a single pulse using the Bio-Rad Gene Pulser (Bio-Rad Laboratories) set at 2.5 kV, 25 μF, with the pulse controller resistance set at infinity.

Article Title: Characterization of an endogenous gene expressed in Aedes aegypti using an orally infectious recombinant Sindbis virus
Article Snippet: Plasmid DNA was linearized by restriction digest with 3–4 fold excess of Xho I. .. This RNA was electroporated into 5 × 106 BHK-21 (baby hamster kidney) cells using a BioRad Gene Pulser set at 450 V, 125 µF, for 0.9 s. Cells and debris from the electroporation were immediately added to 4 ml of Leibovitz L-15 medium (Gibco BRL) supplemented with 10% fetal bovine serum in 25 cm2 tissue culture flasks.

Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae
Article Snippet: All gene targeting, gene cloning and plasmid propagation procedures were performed in E. coli DH10B [Invitrogen, genotype: F- mcr A Δ(mrr -hsd RMS-mcr BC) Φ80lac ZΔM15 Δlac X74 rec A1 end A1 ara D139 Δ(ara leu ) 7697 gal U gal K rps L nup G λ-], incubating plates and liquid cultures at 37°C. .. All oligonucleotides, linear dsDNA and plasmid DNA were transformed into E. coli cells by electroporation using a MicroPulser electroporator with 1 mm gap electroporation cuvettes (BioRad). .. Transformed cells were plated onto Luria-Bertani (LB) agar (USB), and liquid cultures were grown in LB medium (USB); supplementing with kanamycin (Kan, 50 μg/ml, USB), ampicillin (Amp, 50 μg/ml, USB) and/or chloramphenicol (Cm, 30 μg/ml, Sigma) for plasmid maintenance, where appropriate.

Article Title: Construction of a Fully Retargeted Herpes Simplex Virus 1 Recombinant Capable of Entering Cells Solely via Human Epidermal Growth Factor Receptor 2
Article Snippet: The procedure applied to generate recombinant genomes in E. coli was essentially as described previously, with slight modifications ( , , ). .. Briefly, electrocompetent E. coli DH10B harboring the relevant gD-minus HSV-BAC genomes was electroporated with the shuttle vector in 0.2-cm electroporation cuvettes (Bio-Rad) at 200 Ω, 25 μF, and 2.5 kV; plated on LB agar containing 25 μg/ml Kan (the shuttle vector's marker) and 20 μg/ml Cam (the BAC's marker); and incubated at 30°C overnight to allow the expression of RecA from the shuttle vector. .. The clones were replated onto LB plus Kan plus Cam at 43°C to allow the identification of those harboring the cointegrates (visible as large colonies compared to the temperature-sensitive “small-colony” phenotype determined by nonintegrated shuttle vectors).

Article Title: Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes
Article Snippet: Paragraph title: 2.2. Cloning of PCR Fragments into a pUC18 Plasmid ... Electroporation cuvettes (Bio-Rad).

Article Title: Deletion of murine kininogen gene 1 (mKng1) causes loss of plasma kininogen and delays thrombosis
Article Snippet: Briefly, cuvettes containing 0.8 mL cells at a concentration of 7 × 106 cells/mL and 40 μg of linearized DNA were pulsed at 0.240 kV and 500 μF using a Bio-Rad gene pulser (Bio-Rad, Cupertino, CA). .. The screening strategy for evaluating homologous recombination was based on the fact that homologous recombination of the targeting construct with chromosomal DNA within the kininogen gene locus caused changes in the DNA digestion profile that were detectable by Southern blotting using a 5′ external probe and a 3′ internal probe.

Article Title: Biocontrol of the Sugarcane Borer Eldana saccharina by Expression of the Bacillus thuringiensis cry1Ac7 and Serratia marcescens chiA Genes in Sugarcane-Associated Bacteria
Article Snippet: Broad-host-range plasmids and the integration vector carrying the p tac-cry1Ac7 cassette were electroporated into P. fluorescens 14 and H. seropedicae Nal1 using a modification of the method of Waalwijk et al. ( ). .. Cells harvested at mid-exponential phase and washed three times in 300 mM sucrose were electroporated in 40-μl volumes with 1 to 3 μg of DNA using a Bio-Rad Gene Pulser and controller set at 25 μF, 2.5 kV, and 200 Ω. Phenotypic expression was carried out at 30°C for 2 h in 1 ml of LB or JNFb medium (for P. fluorescens and H. seropedicae , respectively).

Article Title: A Novel Testicular RhoGAP-Domain Protein Induces Apoptosis
Article Snippet: Briefly, the complete tGAP1 coding region (sense orientation; 3879 base-pair [bp] Kpn I/ Not I restriction fragment), partial tGAP1 antisense orientation (3166-bp Xho I/ Sal I restriction fragment), and human p53 (1191-bp Xba I/ Bgl II restriction fragment) were subcloned into the pAdTrack-CMV shuttle vector. .. The shuttle-vector plasmids and a shuttle vector without insert were linearized with Pme I and transformed into Escherichia coli strain BJ5183 by electroporation in a Bio-Rad Gene Pulser electroporator (Bio-Rad, Mississauga, ON, Canada): BJ5183 stably contains the adenoviral backbone vector pAdEasy-1. .. Bacterial colonies containing recombinants were selected with kanamycin (50 μg/ml) and screened by restriction endonuclease digestions ( Pac I, Eco RI, and Bam HI).

Software:

Article Title: Roles of the recJ and recN Genes in Homologous Recombination and DNA Repair Pathways of Neisseria gonorrhoeae
Article Snippet: E. coli strains were transformed by electroporation using the Gene Pulser II electroporation system (Bio-Rad Laboratories) as specified by the manufacturer. .. Sequencing reactions were performed using the Big-Dye Terminator cycle-sequencing kit (Perkin-Elmer Corp.), and sequencing products were separated on an ABI model 377 automated DNA sequencer.

Negative Control:

Article Title: Targeting Tryptophan Decarboxylase to Selected Subcellular Compartments of Tobacco Plants Affects Enzyme Stability and in Vivo Function and Leads to a Lesion-Mimic Phenotype
Article Snippet: The plant expression vectors were introduced into A. tumefaciens GV3101 cells by electroporation using a Gene Pulser II electroporation system (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. .. Four young leaves (approximately 6–12 cm in length) per targeting cassette were randomly selected from different plants, infiltrated, and incubated in sealed trays on wet paper (Whatman, Clifton, NJ) at 25°C with a 16-h photoperiod for 60 h. At the end of the incubation, the leaves were weighed, frozen in liquid nitrogen, and stored at −80°C until analyzed.

Recombinant:

Article Title: Characterization of an endogenous gene expressed in Aedes aegypti using an orally infectious recombinant Sindbis virus
Article Snippet: This RNA was electroporated into 5 × 106 BHK-21 (baby hamster kidney) cells using a BioRad Gene Pulser set at 450 V, 125 µF, for 0.9 s. Cells and debris from the electroporation were immediately added to 4 ml of Leibovitz L-15 medium (Gibco BRL) supplemented with 10% fetal bovine serum in 25 cm2 tissue culture flasks. .. Approximately 5–8 × 107 plaque forming units or 7.2–8 log10 TCID50 per ml of MRE/2'3J, MRE/2'3J/ppA, and MRE/2'3J/ppC viruses were obtained.

Article Title: Targeting Tryptophan Decarboxylase to Selected Subcellular Compartments of Tobacco Plants Affects Enzyme Stability and in Vivo Function and Leads to a Lesion-Mimic Phenotype
Article Snippet: The plant expression vectors were introduced into A. tumefaciens GV3101 cells by electroporation using a Gene Pulser II electroporation system (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. .. Tobacco leaves infiltrated with nonrecombinant Agrobacteria were used as negative control.

Article Title: Construction of a Fully Retargeted Herpes Simplex Virus 1 Recombinant Capable of Entering Cells Solely via Human Epidermal Growth Factor Receptor 2
Article Snippet: Paragraph title: Generation of recombinant genomes by two-step replacement in bacteria. ... Briefly, electrocompetent E. coli DH10B harboring the relevant gD-minus HSV-BAC genomes was electroporated with the shuttle vector in 0.2-cm electroporation cuvettes (Bio-Rad) at 200 Ω, 25 μF, and 2.5 kV; plated on LB agar containing 25 μg/ml Kan (the shuttle vector's marker) and 20 μg/ml Cam (the BAC's marker); and incubated at 30°C overnight to allow the expression of RecA from the shuttle vector.

Article Title: A Novel Testicular RhoGAP-Domain Protein Induces Apoptosis
Article Snippet: The shuttle-vector plasmids and a shuttle vector without insert were linearized with Pme I and transformed into Escherichia coli strain BJ5183 by electroporation in a Bio-Rad Gene Pulser electroporator (Bio-Rad, Mississauga, ON, Canada): BJ5183 stably contains the adenoviral backbone vector pAdEasy-1. .. The shuttle-vector plasmids and a shuttle vector without insert were linearized with Pme I and transformed into Escherichia coli strain BJ5183 by electroporation in a Bio-Rad Gene Pulser electroporator (Bio-Rad, Mississauga, ON, Canada): BJ5183 stably contains the adenoviral backbone vector pAdEasy-1.

Agarose Gel Electrophoresis:

Article Title: Overexpression of Lactobacillus caseid-Hydroxyisocaproic Acid Dehydrogenase in Cheddar Cheese
Article Snippet: Transformants were selected on Luria-Bertani agar that contained 500 μg of ERY per ml, plasmid DNA was isolated from Eryr CFU by the alkaline lysis method , and the presence of dhic insert DNA was confirmed by agarose gel electrophoresis and DNA sequence analysis. .. Three microliters of pHADH or pTRKH2 was mixed with 100 μl of cell suspension in a 0.2-cm electroporation cuvette and placed on ice for 3 min. An electric pulse was delivered in a Bio-Rad Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) set to the following parameters: 2.5 kV, 25 μF, and 200 Ω.

Article Title: Characterization of an endogenous gene expressed in Aedes aegypti using an orally infectious recombinant Sindbis virus
Article Snippet: Complete digestion of the DNA was confirmed by agarose gel electrophoresis. .. This RNA was electroporated into 5 × 106 BHK-21 (baby hamster kidney) cells using a BioRad Gene Pulser set at 450 V, 125 µF, for 0.9 s. Cells and debris from the electroporation were immediately added to 4 ml of Leibovitz L-15 medium (Gibco BRL) supplemented with 10% fetal bovine serum in 25 cm2 tissue culture flasks.

In Vitro:

Article Title: Characterization of an endogenous gene expressed in Aedes aegypti using an orally infectious recombinant Sindbis virus
Article Snippet: DNA was transcribed in vitro from the SP6 promoter, and RNA capping was achieved by adding a capping analog (Ambion, Inc.) to the mixture. .. This RNA was electroporated into 5 × 106 BHK-21 (baby hamster kidney) cells using a BioRad Gene Pulser set at 450 V, 125 µF, for 0.9 s. Cells and debris from the electroporation were immediately added to 4 ml of Leibovitz L-15 medium (Gibco BRL) supplemented with 10% fetal bovine serum in 25 cm2 tissue culture flasks.

Transgenic Assay:

Article Title: Introducing a Null Mutation in the Mouse K6? and K6? Genes Reveals Their Essential Structural Role in the Oral Mucosa
Article Snippet: Paragraph title: Construction of Targeting Vector and Generation of Transgenic Mice ... DNA (20 μg) was transfected by electroporation into 2 × 107 exponentially growing 129/Sv CK35 embryonic stem (ES) cells ( ) using a BioRad Gene Pulser apparatus as described ( ).

Article Title: Targeting Tryptophan Decarboxylase to Selected Subcellular Compartments of Tobacco Plants Affects Enzyme Stability and in Vivo Function and Leads to a Lesion-Mimic Phenotype
Article Snippet: The plant expression vectors were introduced into A. tumefaciens GV3101 cells by electroporation using a Gene Pulser II electroporation system (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. .. Tobacco leaves infiltrated with nonrecombinant Agrobacteria were used as negative control.

Article Title: Hydra Mesoglea Proteome Identifies Thrombospondin as a Conserved Component Active in Head Organizer Restriction
Article Snippet: In brief, animals from a daily-fed culture of chimeric ecto-GFP/endo-RFP transgenic animals were washed twice with ultrapure water. .. For each reaction, 20 animals were transferred to electroporation cuvettes (4 mm gap, Bio-Rad) and excess liquid was removed.

Article Title: Deletion of murine kininogen gene 1 (mKng1) causes loss of plasma kininogen and delays thrombosis
Article Snippet: pmKng1 Δ E10 was electroporated into murine ES cells by the transgenic and targeting service of the Department of Genetics, University Hospitals Research Institute (Cleveland, OH). .. Briefly, cuvettes containing 0.8 mL cells at a concentration of 7 × 106 cells/mL and 40 μg of linearized DNA were pulsed at 0.240 kV and 500 μF using a Bio-Rad gene pulser (Bio-Rad, Cupertino, CA).

Functional Assay:

Article Title: Introducing a Null Mutation in the Mouse K6? and K6? Genes Reveals Their Essential Structural Role in the Oral Mucosa
Article Snippet: Plasmid NeoTKXho, with functional PGK-Neo (Neo) and MC1TK (TK [thymidine kinase]) cassettes for selection, was obtained from Dr. Jeremy Nathans (Johns Hopkins University, Baltimore, MD) and used to generate the targeting vector. .. DNA (20 μg) was transfected by electroporation into 2 × 107 exponentially growing 129/Sv CK35 embryonic stem (ES) cells ( ) using a BioRad Gene Pulser apparatus as described ( ).

Produced:

Article Title: Properties of the Naturally Occurring Soluble Surface Glycoprotein of Ecotropic Murine Leukemia Virus: Binding Specificity and Possible Conformational Change after Binding to Receptor
Article Snippet: SIRC-NIH EcoR cells were established by transfection of SIRC cells with pcDNA-NIH EcoR (see below) by the electroporation method (Gene Pulser; Bio-Rad), and transformants were selected in a medium containing 1 mg of Geneticin (Sigma) per ml. .. NIH 3T3-HmFCR cells were NIH 3T3 cells transfected with a plasmid, pHmFCR , with Lipofectamine reagent (Gibco BRL) and were selected with 100 μg of hygromycin-B (Wako Pure Chemical Industries, Ltd.) per ml.

Concentration Assay:

Article Title: Deletion of murine kininogen gene 1 (mKng1) causes loss of plasma kininogen and delays thrombosis
Article Snippet: pmKng1 Δ E10 was electroporated into murine ES cells by the transgenic and targeting service of the Department of Genetics, University Hospitals Research Institute (Cleveland, OH). .. Briefly, cuvettes containing 0.8 mL cells at a concentration of 7 × 106 cells/mL and 40 μg of linearized DNA were pulsed at 0.240 kV and 500 μF using a Bio-Rad gene pulser (Bio-Rad, Cupertino, CA). .. After 24 hours, medium containing G418 (200 μg/mL) was added to the cells and changed daily.

Alkaline Lysis:

Article Title: Overexpression of Lactobacillus caseid-Hydroxyisocaproic Acid Dehydrogenase in Cheddar Cheese
Article Snippet: Transformants were selected on Luria-Bertani agar that contained 500 μg of ERY per ml, plasmid DNA was isolated from Eryr CFU by the alkaline lysis method , and the presence of dhic insert DNA was confirmed by agarose gel electrophoresis and DNA sequence analysis. .. Three microliters of pHADH or pTRKH2 was mixed with 100 μl of cell suspension in a 0.2-cm electroporation cuvette and placed on ice for 3 min. An electric pulse was delivered in a Bio-Rad Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) set to the following parameters: 2.5 kV, 25 μF, and 200 Ω.

Article Title: Participation of fad and mbt Genes in Synthesis of Mycobactin in Mycobacterium smegmatis
Article Snippet: Isolation of plasmid DNA from M. smegmatis ( ) was essentially like that from E. coli , i.e., alkaline lysis, except that the cells were first disrupted (beaten for 2 min) by glass beads in a Mini-BeadBeater (Biospec Products, Bartlesville, Okla.). .. Bacteria suspended in 10% glycerol electroporation buffer with 1 μg of DNA were subjected to a single pulse using the Bio-Rad Gene Pulser (Bio-Rad Laboratories) set at 2.5 kV, 25 μF, with the pulse controller resistance set at infinity.

BAC Assay:

Article Title: Construction of a Fully Retargeted Herpes Simplex Virus 1 Recombinant Capable of Entering Cells Solely via Human Epidermal Growth Factor Receptor 2
Article Snippet: The procedure applied to generate recombinant genomes in E. coli was essentially as described previously, with slight modifications ( , , ). .. Briefly, electrocompetent E. coli DH10B harboring the relevant gD-minus HSV-BAC genomes was electroporated with the shuttle vector in 0.2-cm electroporation cuvettes (Bio-Rad) at 200 Ω, 25 μF, and 2.5 kV; plated on LB agar containing 25 μg/ml Kan (the shuttle vector's marker) and 20 μg/ml Cam (the BAC's marker); and incubated at 30°C overnight to allow the expression of RecA from the shuttle vector. .. The clones were replated onto LB plus Kan plus Cam at 43°C to allow the identification of those harboring the cointegrates (visible as large colonies compared to the temperature-sensitive “small-colony” phenotype determined by nonintegrated shuttle vectors).

Marker:

Article Title: Construction of a Fully Retargeted Herpes Simplex Virus 1 Recombinant Capable of Entering Cells Solely via Human Epidermal Growth Factor Receptor 2
Article Snippet: The procedure applied to generate recombinant genomes in E. coli was essentially as described previously, with slight modifications ( , , ). .. Briefly, electrocompetent E. coli DH10B harboring the relevant gD-minus HSV-BAC genomes was electroporated with the shuttle vector in 0.2-cm electroporation cuvettes (Bio-Rad) at 200 Ω, 25 μF, and 2.5 kV; plated on LB agar containing 25 μg/ml Kan (the shuttle vector's marker) and 20 μg/ml Cam (the BAC's marker); and incubated at 30°C overnight to allow the expression of RecA from the shuttle vector. .. The clones were replated onto LB plus Kan plus Cam at 43°C to allow the identification of those harboring the cointegrates (visible as large colonies compared to the temperature-sensitive “small-colony” phenotype determined by nonintegrated shuttle vectors).

Chick Chorioallantoic Membrane Assay:

Article Title: Construction of a Fully Retargeted Herpes Simplex Virus 1 Recombinant Capable of Entering Cells Solely via Human Epidermal Growth Factor Receptor 2
Article Snippet: The procedure applied to generate recombinant genomes in E. coli was essentially as described previously, with slight modifications ( , , ). .. Briefly, electrocompetent E. coli DH10B harboring the relevant gD-minus HSV-BAC genomes was electroporated with the shuttle vector in 0.2-cm electroporation cuvettes (Bio-Rad) at 200 Ω, 25 μF, and 2.5 kV; plated on LB agar containing 25 μg/ml Kan (the shuttle vector's marker) and 20 μg/ml Cam (the BAC's marker); and incubated at 30°C overnight to allow the expression of RecA from the shuttle vector. .. The clones were replated onto LB plus Kan plus Cam at 43°C to allow the identification of those harboring the cointegrates (visible as large colonies compared to the temperature-sensitive “small-colony” phenotype determined by nonintegrated shuttle vectors).

Homologous Recombination:

Article Title: Deletion of murine kininogen gene 1 (mKng1) causes loss of plasma kininogen and delays thrombosis
Article Snippet: Briefly, cuvettes containing 0.8 mL cells at a concentration of 7 × 106 cells/mL and 40 μg of linearized DNA were pulsed at 0.240 kV and 500 μF using a Bio-Rad gene pulser (Bio-Rad, Cupertino, CA). .. Briefly, cuvettes containing 0.8 mL cells at a concentration of 7 × 106 cells/mL and 40 μg of linearized DNA were pulsed at 0.240 kV and 500 μF using a Bio-Rad gene pulser (Bio-Rad, Cupertino, CA).

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    Bio-Rad gap electroporation cuvette
    The mice before treatment on the top and the same mice after the treatment on the bottom, where ( A ) treated with distilled water; ( B ) treated with distilled water and <t>electroporation</t> (25 kV/cm × 900 ns × 1000 pulses); ( C ) treated with acetic acid 1%; ( D ) treated with acetic acid 1% and electroporation. Combined treatment allows full eradication of bacteria.
    Gap Electroporation Cuvette, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The mice before treatment on the top and the same mice after the treatment on the bottom, where ( A ) treated with distilled water; ( B ) treated with distilled water and electroporation (25 kV/cm × 900 ns × 1000 pulses); ( C ) treated with acetic acid 1%; ( D ) treated with acetic acid 1% and electroporation. Combined treatment allows full eradication of bacteria.

    Journal: Scientific Reports

    Article Title: Non-invasive nanosecond electroporation for biocontrol of surface infections: an in vivo study

    doi: 10.1038/s41598-018-32783-7

    Figure Lengend Snippet: The mice before treatment on the top and the same mice after the treatment on the bottom, where ( A ) treated with distilled water; ( B ) treated with distilled water and electroporation (25 kV/cm × 900 ns × 1000 pulses); ( C ) treated with acetic acid 1%; ( D ) treated with acetic acid 1% and electroporation. Combined treatment allows full eradication of bacteria.

    Article Snippet: For PEF permeabilization, the 60 μl of the resultant suspension was transferred to 1 mm gap electroporation cuvette (Biorad, Hercules, USA).

    Techniques: Mouse Assay, Electroporation

    The example of fluorescence photographs of propidium iodide negative ( A ) and propidium iodide positive ( B ) cells. For better perception and indication of permeabilization multiple cells are shown, however for quantitative analysis of cell permeabilization only single cells were used. Due to electroporation ( B ) the propidium iodide can successfully enter the cells, which is not the case in the untreated control ( A ). Ch01 – brightfield image; PI – fluorescence image (488 nm), taken using bandpass filter of 610–630 nm.

    Journal: Scientific Reports

    Article Title: Non-invasive nanosecond electroporation for biocontrol of surface infections: an in vivo study

    doi: 10.1038/s41598-018-32783-7

    Figure Lengend Snippet: The example of fluorescence photographs of propidium iodide negative ( A ) and propidium iodide positive ( B ) cells. For better perception and indication of permeabilization multiple cells are shown, however for quantitative analysis of cell permeabilization only single cells were used. Due to electroporation ( B ) the propidium iodide can successfully enter the cells, which is not the case in the untreated control ( A ). Ch01 – brightfield image; PI – fluorescence image (488 nm), taken using bandpass filter of 610–630 nm.

    Article Snippet: For PEF permeabilization, the 60 μl of the resultant suspension was transferred to 1 mm gap electroporation cuvette (Biorad, Hercules, USA).

    Techniques: Fluorescence, Electroporation

    The dependence of P . aeruginosa luminescence on the applied treatment parameters, live bacteria luminesce; the width of colored area corresponds to standard deviation of data during selected treatment protocol; PEF – 25 kV/cm × 900 ns × 1000 pulses; AA – acetic acid. The 1% acetic acid combined with electroporation allowed complete inactivation of bacteria in vitro , which is unachievable if the treatments are used separately.

    Journal: Scientific Reports

    Article Title: Non-invasive nanosecond electroporation for biocontrol of surface infections: an in vivo study

    doi: 10.1038/s41598-018-32783-7

    Figure Lengend Snippet: The dependence of P . aeruginosa luminescence on the applied treatment parameters, live bacteria luminesce; the width of colored area corresponds to standard deviation of data during selected treatment protocol; PEF – 25 kV/cm × 900 ns × 1000 pulses; AA – acetic acid. The 1% acetic acid combined with electroporation allowed complete inactivation of bacteria in vitro , which is unachievable if the treatments are used separately.

    Article Snippet: For PEF permeabilization, the 60 μl of the resultant suspension was transferred to 1 mm gap electroporation cuvette (Biorad, Hercules, USA).

    Techniques: Standard Deviation, Electroporation, In Vitro

    The in vivo potential of acetic acid and electroporation for inactivation of P . aeruginosa . The combined treatment (AA 1% and PEF) results in up to full eradication of bacteria, where PEF: 25 kV/cm × 900 ns × 1000 pulses delivered at pulse repetition frequency of 15 kHz.

    Journal: Scientific Reports

    Article Title: Non-invasive nanosecond electroporation for biocontrol of surface infections: an in vivo study

    doi: 10.1038/s41598-018-32783-7

    Figure Lengend Snippet: The in vivo potential of acetic acid and electroporation for inactivation of P . aeruginosa . The combined treatment (AA 1% and PEF) results in up to full eradication of bacteria, where PEF: 25 kV/cm × 900 ns × 1000 pulses delivered at pulse repetition frequency of 15 kHz.

    Article Snippet: For PEF permeabilization, the 60 μl of the resultant suspension was transferred to 1 mm gap electroporation cuvette (Biorad, Hercules, USA).

    Techniques: In Vivo, Electroporation

    The schematic representation of proposed electroporation mediated methodology for biocontrol of surface infections, where PEF – pulsed electric field; nsPEF – high frequency nanosecond PEF bursts. Distilled water or low concentrations of acetic acid were used as an electrode-skin interface.

    Journal: Scientific Reports

    Article Title: Non-invasive nanosecond electroporation for biocontrol of surface infections: an in vivo study

    doi: 10.1038/s41598-018-32783-7

    Figure Lengend Snippet: The schematic representation of proposed electroporation mediated methodology for biocontrol of surface infections, where PEF – pulsed electric field; nsPEF – high frequency nanosecond PEF bursts. Distilled water or low concentrations of acetic acid were used as an electrode-skin interface.

    Article Snippet: For PEF permeabilization, the 60 μl of the resultant suspension was transferred to 1 mm gap electroporation cuvette (Biorad, Hercules, USA).

    Techniques: Electroporation

    Differentiation potential of hAD-MSCs and fibroblasts following transfection using four independent techniques. Osteogenic, adipogenic, and chondrogenic differentiation of ( A ) hAD-MSCs and ( B ) fibroblasts post-transfection using the microporation, standard electroporation, cationic polymer, and classical calcium phosphate precipitation transfection techniques. After 21 days, osteogenic cultures were stained using alizarin red. Adipogenic cultures were analyzed for lipid accumulation after 14 days of differentiation using inverted microscopy. Thereafter they were fixed and stained for triglycerides with Oil-Red-O. After 21 days, chondrogenic cultures were stained for spheroid formation using Alcian blue. (Magnification: ( A ) adipogenic 400×, osteogenic 40× and chondrogenic 40×; ( B ) adipogenic 400×, osteogenic 100× and chondrogenic 100×).

    Journal: International Journal of Molecular Sciences

    Article Title: A Comparative Study of Non-Viral Gene Delivery Techniques to Human Adipose-Derived Mesenchymal Stem Cell

    doi: 10.3390/ijms150915044

    Figure Lengend Snippet: Differentiation potential of hAD-MSCs and fibroblasts following transfection using four independent techniques. Osteogenic, adipogenic, and chondrogenic differentiation of ( A ) hAD-MSCs and ( B ) fibroblasts post-transfection using the microporation, standard electroporation, cationic polymer, and classical calcium phosphate precipitation transfection techniques. After 21 days, osteogenic cultures were stained using alizarin red. Adipogenic cultures were analyzed for lipid accumulation after 14 days of differentiation using inverted microscopy. Thereafter they were fixed and stained for triglycerides with Oil-Red-O. After 21 days, chondrogenic cultures were stained for spheroid formation using Alcian blue. (Magnification: ( A ) adipogenic 400×, osteogenic 40× and chondrogenic 40×; ( B ) adipogenic 400×, osteogenic 100× and chondrogenic 100×).

    Article Snippet: A total of 200 μL of cells were mixed with 10 μg DNA transferred to a 4 mm electroporation cuvette (BioRad, Hercules, CA, USA), and electroporated using Gene Pulser Xcell electroporation system (BioRad).

    Techniques: Transfection, Electroporation, Staining, Inverted Microscopy

    Transfection efficiencies of hAD-MSCs and fibroblasts using four independent techniques. The microporation technique had the highest transfection efficiency as compared to standard electroporation and chemical-based transfection reagents for transfection of ( A ) hAD-MSCs and ( B ) fibroblasts. hAD-MSCs and fibroblasts were transfected with a plasmid encoding for Angiopoietin-1 (ANGPT-1) and the enhanced green fluorescent protein (eGFP) expression cassette using different chemical-based reagents and electroporation protocols after optimization.

    Journal: International Journal of Molecular Sciences

    Article Title: A Comparative Study of Non-Viral Gene Delivery Techniques to Human Adipose-Derived Mesenchymal Stem Cell

    doi: 10.3390/ijms150915044

    Figure Lengend Snippet: Transfection efficiencies of hAD-MSCs and fibroblasts using four independent techniques. The microporation technique had the highest transfection efficiency as compared to standard electroporation and chemical-based transfection reagents for transfection of ( A ) hAD-MSCs and ( B ) fibroblasts. hAD-MSCs and fibroblasts were transfected with a plasmid encoding for Angiopoietin-1 (ANGPT-1) and the enhanced green fluorescent protein (eGFP) expression cassette using different chemical-based reagents and electroporation protocols after optimization.

    Article Snippet: A total of 200 μL of cells were mixed with 10 μg DNA transferred to a 4 mm electroporation cuvette (BioRad, Hercules, CA, USA), and electroporated using Gene Pulser Xcell electroporation system (BioRad).

    Techniques: Transfection, Electroporation, Plasmid Preparation, Expressing

    Post-transfection proliferative potential of hAD-MSCs and fibroblasts. Analysis of ( A ) hAD-MSC and ( B ) fibroblast proliferation capability following transfection using microporation, standard electroporation, cationic polymer, and classical calcium phosphate precipitation transfection techniques. Two days post-transfection, the cells were trypsinized and seeded at 3 × 10 3 cells/well in 96-well plates. The Trypan blue exclusion assay was used to determine total cell number in the hAD-MSC and fibroblast cultures at days 3, 5, and 7 after plating. hAD-MSCs and fibroblasts transfected using the microporation, standard electroporation, and classical calcium phosphate precipitation techniques exhibited a greater proliferation potential comparable to non-transfected cells. However, transfection with the cationic polymer repressed the hAD-MSCs and fibroblasts proliferation.

    Journal: International Journal of Molecular Sciences

    Article Title: A Comparative Study of Non-Viral Gene Delivery Techniques to Human Adipose-Derived Mesenchymal Stem Cell

    doi: 10.3390/ijms150915044

    Figure Lengend Snippet: Post-transfection proliferative potential of hAD-MSCs and fibroblasts. Analysis of ( A ) hAD-MSC and ( B ) fibroblast proliferation capability following transfection using microporation, standard electroporation, cationic polymer, and classical calcium phosphate precipitation transfection techniques. Two days post-transfection, the cells were trypsinized and seeded at 3 × 10 3 cells/well in 96-well plates. The Trypan blue exclusion assay was used to determine total cell number in the hAD-MSC and fibroblast cultures at days 3, 5, and 7 after plating. hAD-MSCs and fibroblasts transfected using the microporation, standard electroporation, and classical calcium phosphate precipitation techniques exhibited a greater proliferation potential comparable to non-transfected cells. However, transfection with the cationic polymer repressed the hAD-MSCs and fibroblasts proliferation.

    Article Snippet: A total of 200 μL of cells were mixed with 10 μg DNA transferred to a 4 mm electroporation cuvette (BioRad, Hercules, CA, USA), and electroporated using Gene Pulser Xcell electroporation system (BioRad).

    Techniques: Transfection, Electroporation, Trypan Blue Exclusion Assay

    Time-course analysis of eGFP expression level in hAD-MSCs and fibroblasts. Analysis of eGFP expression of ( A ) hAD-MSCs and ( B ) fibroblasts following transfection using the microporation, standard electroporation, cationic polymer, and classical calcium phosphate precipitation techniques over a 21-day period (Magnification: ( A ) 100× and ( B ) 100×); ( C ) Evaluation of eGFP expression in microporated hAD-MSCs and fibroblasts over an 8-day period. The expression of eGFP was detected by fluorescence microscopy at days 2, 4, 6, and 8 following transfection. The number of eGFP-expressing cells began to decline over time.

    Journal: International Journal of Molecular Sciences

    Article Title: A Comparative Study of Non-Viral Gene Delivery Techniques to Human Adipose-Derived Mesenchymal Stem Cell

    doi: 10.3390/ijms150915044

    Figure Lengend Snippet: Time-course analysis of eGFP expression level in hAD-MSCs and fibroblasts. Analysis of eGFP expression of ( A ) hAD-MSCs and ( B ) fibroblasts following transfection using the microporation, standard electroporation, cationic polymer, and classical calcium phosphate precipitation techniques over a 21-day period (Magnification: ( A ) 100× and ( B ) 100×); ( C ) Evaluation of eGFP expression in microporated hAD-MSCs and fibroblasts over an 8-day period. The expression of eGFP was detected by fluorescence microscopy at days 2, 4, 6, and 8 following transfection. The number of eGFP-expressing cells began to decline over time.

    Article Snippet: A total of 200 μL of cells were mixed with 10 μg DNA transferred to a 4 mm electroporation cuvette (BioRad, Hercules, CA, USA), and electroporated using Gene Pulser Xcell electroporation system (BioRad).

    Techniques: Expressing, Transfection, Electroporation, Fluorescence, Microscopy

    Uptake of cy3-labeled DNA into isolated mitochondria upon electroporation: ( Top ) Schematic representation of the experiment; and ( Bottom ) ( left ) phase contrast images of purified mitochondria incubated with a 847 bp cy3-labeled PCR fragment without (0 kV/cm) or with electroporation at either 10 kV/cm or 13 kV/cm; ( center ) visualization of cy3 fragment uptake via fluorescence microscopy; and ( right ) overlay of the fluorescent signals (shown in color) onto the respective phase images.

    Journal: Genes

    Article Title: Electroporation of DNA into Physarum polycephalum Mitochondria: Effects on Transcription and RNA Editing in Isolated Organelles

    doi: 10.3390/genes7120128

    Figure Lengend Snippet: Uptake of cy3-labeled DNA into isolated mitochondria upon electroporation: ( Top ) Schematic representation of the experiment; and ( Bottom ) ( left ) phase contrast images of purified mitochondria incubated with a 847 bp cy3-labeled PCR fragment without (0 kV/cm) or with electroporation at either 10 kV/cm or 13 kV/cm; ( center ) visualization of cy3 fragment uptake via fluorescence microscopy; and ( right ) overlay of the fluorescent signals (shown in color) onto the respective phase images.

    Article Snippet: Mitochondria were then transferred to a cold 0.1 cm gap electroporation cuvette (Bio Rad) and electroporated using an Eppendorf Electroporator 2510 (Hauppauge, NY, USA) at 10 μF, 600 Ω, and field strengths between 5 kV/cm and 20 kV/cm.

    Techniques: Labeling, Isolation, Electroporation, Purification, Incubation, Polymerase Chain Reaction, Fluorescence, Microscopy

    The editing machinery is still functional after electroporation. Gel electrophoresis of RNase T1 products generated from S1 nuclease-protected RNA fragments that encompass the last seven editing sites of the atpA mRNA. Internally labeled RNAs were synthesized by mitochondria before electroporation (M−, lane 3), mitochondria after electroporation in the absence of exogenous DNA (M+, lanes 4 and 9), and mitochondria after electroporation in the presence of exogenous DNA (M +D, lanes 5 and 10). Different amounts of the same RNase T1 digests were loaded in lanes 4 and 9 and lanes 5 and 10. Multiple dilutions of RNase T1 digests of S1 nuclease-protected unedited (Un, lanes 1, 6, and 11) and edited (Ed, lanes 2, 7, and 8) control transcripts were run alongside for comparison. The sequence of the RNase T1 fragments from the protected region of the atpA mRNA is shown at the right, with the C residue added at editing sites 48 through 54 (es48–es54) indicated by a lower case c . Easily resolvable RNase T1 fragments diagnostic of editing are underlined and the size in nucleotides (nts) of the edited and unedited fragments are indicated on the sequence and to the left of the gel.

    Journal: Genes

    Article Title: Electroporation of DNA into Physarum polycephalum Mitochondria: Effects on Transcription and RNA Editing in Isolated Organelles

    doi: 10.3390/genes7120128

    Figure Lengend Snippet: The editing machinery is still functional after electroporation. Gel electrophoresis of RNase T1 products generated from S1 nuclease-protected RNA fragments that encompass the last seven editing sites of the atpA mRNA. Internally labeled RNAs were synthesized by mitochondria before electroporation (M−, lane 3), mitochondria after electroporation in the absence of exogenous DNA (M+, lanes 4 and 9), and mitochondria after electroporation in the presence of exogenous DNA (M +D, lanes 5 and 10). Different amounts of the same RNase T1 digests were loaded in lanes 4 and 9 and lanes 5 and 10. Multiple dilutions of RNase T1 digests of S1 nuclease-protected unedited (Un, lanes 1, 6, and 11) and edited (Ed, lanes 2, 7, and 8) control transcripts were run alongside for comparison. The sequence of the RNase T1 fragments from the protected region of the atpA mRNA is shown at the right, with the C residue added at editing sites 48 through 54 (es48–es54) indicated by a lower case c . Easily resolvable RNase T1 fragments diagnostic of editing are underlined and the size in nucleotides (nts) of the edited and unedited fragments are indicated on the sequence and to the left of the gel.

    Article Snippet: Mitochondria were then transferred to a cold 0.1 cm gap electroporation cuvette (Bio Rad) and electroporated using an Eppendorf Electroporator 2510 (Hauppauge, NY, USA) at 10 μF, 600 Ω, and field strengths between 5 kV/cm and 20 kV/cm.

    Techniques: Functional Assay, Electroporation, Nucleic Acid Electrophoresis, Generated, Labeling, Synthesized, Sequencing, Diagnostic Assay

    Transcription of endogenous genes is not altered by electroporation: ( A ) Transcript map of the genes probed in dot blot experiments using gene designations from [ 7 ]. ( B ) Grid positions of immobilized PCR fragments; ( C ) Dot blots probed with labeled RNA synthesized by: mitochondria before electroporation ( top ); mitochondria after electroporation in the absence of exogenous DNA ( middle ); and mitochondria after electroporation in the presence of exogenous DNA ( bottom ). ( D ) Quantification of the data in ( C ) showing relative expression levels of individual genes under each of the three conditions.

    Journal: Genes

    Article Title: Electroporation of DNA into Physarum polycephalum Mitochondria: Effects on Transcription and RNA Editing in Isolated Organelles

    doi: 10.3390/genes7120128

    Figure Lengend Snippet: Transcription of endogenous genes is not altered by electroporation: ( A ) Transcript map of the genes probed in dot blot experiments using gene designations from [ 7 ]. ( B ) Grid positions of immobilized PCR fragments; ( C ) Dot blots probed with labeled RNA synthesized by: mitochondria before electroporation ( top ); mitochondria after electroporation in the absence of exogenous DNA ( middle ); and mitochondria after electroporation in the presence of exogenous DNA ( bottom ). ( D ) Quantification of the data in ( C ) showing relative expression levels of individual genes under each of the three conditions.

    Article Snippet: Mitochondria were then transferred to a cold 0.1 cm gap electroporation cuvette (Bio Rad) and electroporated using an Eppendorf Electroporator 2510 (Hauppauge, NY, USA) at 10 μF, 600 Ω, and field strengths between 5 kV/cm and 20 kV/cm.

    Techniques: Electroporation, Dot Blot, Polymerase Chain Reaction, Labeling, Synthesized, Expressing

    DNA introduced into mitochondria is transcribed: ( A ) Reverse transcription PCR (RT-PCR) products derived from RNA synthesized after incubation of isolated mitochondria with a 847 bp cy3-labeled PCR fragment with (10 kV/cm, 13 kV/cm) or without (0 kV/cm) electroporation. The +RT/−RT designation indicates reverse transcription reactions done in the presence or absence of enzyme, respectively. Lanes 1 and 10 contain size standards VI and V (Roche), respectively (M); PCR controls are shown in lanes 8 and 9. ( B ) RT-PCR products derived from RNA synthesized after incubation of isolated mitochondria with a 3.9 kb plasmid with (12 kV/cm) or without (0 kV/cm) electroporation. Samples in lanes 6 and 7 demonstrate the lack of background with total mitochondrial RNA (no exogenous DNA, no electroporation) with this set of plasmid-specific PCR primers. Other labels as in A.

    Journal: Genes

    Article Title: Electroporation of DNA into Physarum polycephalum Mitochondria: Effects on Transcription and RNA Editing in Isolated Organelles

    doi: 10.3390/genes7120128

    Figure Lengend Snippet: DNA introduced into mitochondria is transcribed: ( A ) Reverse transcription PCR (RT-PCR) products derived from RNA synthesized after incubation of isolated mitochondria with a 847 bp cy3-labeled PCR fragment with (10 kV/cm, 13 kV/cm) or without (0 kV/cm) electroporation. The +RT/−RT designation indicates reverse transcription reactions done in the presence or absence of enzyme, respectively. Lanes 1 and 10 contain size standards VI and V (Roche), respectively (M); PCR controls are shown in lanes 8 and 9. ( B ) RT-PCR products derived from RNA synthesized after incubation of isolated mitochondria with a 3.9 kb plasmid with (12 kV/cm) or without (0 kV/cm) electroporation. Samples in lanes 6 and 7 demonstrate the lack of background with total mitochondrial RNA (no exogenous DNA, no electroporation) with this set of plasmid-specific PCR primers. Other labels as in A.

    Article Snippet: Mitochondria were then transferred to a cold 0.1 cm gap electroporation cuvette (Bio Rad) and electroporated using an Eppendorf Electroporator 2510 (Hauppauge, NY, USA) at 10 μF, 600 Ω, and field strengths between 5 kV/cm and 20 kV/cm.

    Techniques: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Synthesized, Incubation, Isolation, Labeling, Electroporation, Plasmid Preparation