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Roche rapid dna ligation kit
Schematic representation of the expression constructs ( A ) pLgals1-hum-β3GN-T2 and ( B ) pLgals1-hum-MFng . The <t>PCR-amplified</t> <t>DNA</t> sequences were inserted in the pLgals1-Tev vector construct at the multicloning site, which is preceded by the 6HisTag
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1) Product Images from "The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2"

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2

Journal: Glycobiology

doi: 10.1093/glycob/cwr110

Schematic representation of the expression constructs ( A ) pLgals1-hum-β3GN-T2 and ( B ) pLgals1-hum-MFng . The PCR-amplified DNA sequences were inserted in the pLgals1-Tev vector construct at the multicloning site, which is preceded by the 6HisTag
Figure Legend Snippet: Schematic representation of the expression constructs ( A ) pLgals1-hum-β3GN-T2 and ( B ) pLgals1-hum-MFng . The PCR-amplified DNA sequences were inserted in the pLgals1-Tev vector construct at the multicloning site, which is preceded by the 6HisTag

Techniques Used: Expressing, Construct, Polymerase Chain Reaction, Amplification, Plasmid Preparation

2) Product Images from "Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development"

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development

Journal: PLoS ONE

doi: 10.1371/journal.pone.0028537

Analysis of RAX mRNA expression in the brains of rep mice. Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.
Figure Legend Snippet: Analysis of RAX mRNA expression in the brains of rep mice. Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.

Techniques Used: Expressing, Mouse Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

3) Product Images from "Overexpression of VEGF165b in Podocytes Reduces Glomerular Permeability"

Article Title: Overexpression of VEGF165b in Podocytes Reduces Glomerular Permeability

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2009060617

Generation of pNeph-VEGF 165 b heterozygous transgenic mice. (A) VEGF 165 b is cloned into an expression vector under the control of the Nephrin promoter (Transgenic Construct; Ai). When transfected into human podocytes, VEGF 165 b expression was significantly greater than control vector or untransfected cells (Aii). (B) PCR screen of pups born from injected embryos. (C) Southern blot, confirming single insertion into genomic DNA. The gDNA is digested with EcoRI, and a probe made from the cDNA is used to generate the transgene. There was a single EcoRI site in the 5′ end of the insert, so multiple insertions should generate multiple different-sized bands in the transgene. There is only a single band in line 1, indicating a single insertion point (insertions into multiple sites would generate multiple bands). See Supplemental Figure S1. Each lane in B and C represents a distinct animal line. The mouse nephrin sequence is also detected as seen in the WT animals (two bands as shown by arrows). Line 1 is used for establishment of the subsequent heterozygous and homozygous breeding colonies. (D) Of note, the ultrafiltration fraction from potential founder lines is not different from each other.
Figure Legend Snippet: Generation of pNeph-VEGF 165 b heterozygous transgenic mice. (A) VEGF 165 b is cloned into an expression vector under the control of the Nephrin promoter (Transgenic Construct; Ai). When transfected into human podocytes, VEGF 165 b expression was significantly greater than control vector or untransfected cells (Aii). (B) PCR screen of pups born from injected embryos. (C) Southern blot, confirming single insertion into genomic DNA. The gDNA is digested with EcoRI, and a probe made from the cDNA is used to generate the transgene. There was a single EcoRI site in the 5′ end of the insert, so multiple insertions should generate multiple different-sized bands in the transgene. There is only a single band in line 1, indicating a single insertion point (insertions into multiple sites would generate multiple bands). See Supplemental Figure S1. Each lane in B and C represents a distinct animal line. The mouse nephrin sequence is also detected as seen in the WT animals (two bands as shown by arrows). Line 1 is used for establishment of the subsequent heterozygous and homozygous breeding colonies. (D) Of note, the ultrafiltration fraction from potential founder lines is not different from each other.

Techniques Used: Transgenic Assay, Mouse Assay, Clone Assay, Expressing, Plasmid Preparation, Construct, Transfection, Polymerase Chain Reaction, Injection, Southern Blot, Sequencing

4) Product Images from "A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification"

Article Title: A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

Journal: Journal of Virology

doi: 10.1128/JVI.78.10.4993-4998.2004

Restriction enzyme analysis of the bovine tissue DNA before and after multiply primed RCA. One microliter of the nonamplified DNA, containing 3.9 μg of total DNA, was digested with EcoRI (lane 1). Multiply primed RCA with 450 μM extra dNTPs was performed with 1 μl (3.9 μg) of input bovine tissue DNA, and 2 μl of the RCA product was digested with EcoRI (lane 2), BamHI (lane 3), HindIII (lane 4), SalI (lane 5), and HincII (lane 6). As a negative control, 2 μl of multiply primed RCA product of water was digested with EcoRI (lane 7). Lane M, DNA molecular size marker (Fermentas).
Figure Legend Snippet: Restriction enzyme analysis of the bovine tissue DNA before and after multiply primed RCA. One microliter of the nonamplified DNA, containing 3.9 μg of total DNA, was digested with EcoRI (lane 1). Multiply primed RCA with 450 μM extra dNTPs was performed with 1 μl (3.9 μg) of input bovine tissue DNA, and 2 μl of the RCA product was digested with EcoRI (lane 2), BamHI (lane 3), HindIII (lane 4), SalI (lane 5), and HincII (lane 6). As a negative control, 2 μl of multiply primed RCA product of water was digested with EcoRI (lane 7). Lane M, DNA molecular size marker (Fermentas).

Techniques Used: Negative Control, Marker

5) Product Images from "Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load"

Article Title: Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.00742-14

Establishment of the cvPCR method and the limits of detection. (A) Authentic SFTSV strain HB29 viral RNA (HB29), the plasmid pCR-SFTSV-posicon (P.C.), which contains the artificial sequence, and the nontemplate control (H 2 O) were amplified by cvPCR using primer set 1 or 2. The PCR products were digested by EcoRI (+EcoRI). The sizes of the products were estimated by using a 2-log DNA ladder marker (NEB). (B and C) Isolated strains HB29, YG1, and SPL005, expanded in Vero cells, were diluted with serum from a healthy donor. The purified viral RNAs from the dilutions of virus-spiked serum were amplified. The resulting HB29, YG1, or SPL005 was amplified by primer set 1 (B) or 2 (C). The listed values are the dilutions of the viral strains. NTC, nontemplate control.
Figure Legend Snippet: Establishment of the cvPCR method and the limits of detection. (A) Authentic SFTSV strain HB29 viral RNA (HB29), the plasmid pCR-SFTSV-posicon (P.C.), which contains the artificial sequence, and the nontemplate control (H 2 O) were amplified by cvPCR using primer set 1 or 2. The PCR products were digested by EcoRI (+EcoRI). The sizes of the products were estimated by using a 2-log DNA ladder marker (NEB). (B and C) Isolated strains HB29, YG1, and SPL005, expanded in Vero cells, were diluted with serum from a healthy donor. The purified viral RNAs from the dilutions of virus-spiked serum were amplified. The resulting HB29, YG1, or SPL005 was amplified by primer set 1 (B) or 2 (C). The listed values are the dilutions of the viral strains. NTC, nontemplate control.

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Sequencing, Amplification, Marker, Isolation, Purification

6) Product Images from "Frequency of Spontaneous Mutations in an Avian Hepadnavirus Infection"

Article Title: Frequency of Spontaneous Mutations in an Avian Hepadnavirus Infection

Journal: Journal of Virology

doi: 10.1128/JVI.75.20.9623-9632.2001

PCR assay of the emergence of WT virus in ducklings with mixed infections. Serum samples from individual infected ducks, represented in separate lanes, were amplified by PCR. The products were each mixed with 600 ng of pGEM-7Zf(+) DNA and digested with Sma I to detect the appearance of WT internal reference genomes. The upper band (C) is the control added to each reaction mixture to monitor for complete digestion. The double bands represent the undigested PCR product of G133E-derived genomes (G) and the digested product of WT internal reference genomes (W).
Figure Legend Snippet: PCR assay of the emergence of WT virus in ducklings with mixed infections. Serum samples from individual infected ducks, represented in separate lanes, were amplified by PCR. The products were each mixed with 600 ng of pGEM-7Zf(+) DNA and digested with Sma I to detect the appearance of WT internal reference genomes. The upper band (C) is the control added to each reaction mixture to monitor for complete digestion. The double bands represent the undigested PCR product of G133E-derived genomes (G) and the digested product of WT internal reference genomes (W).

Techniques Used: Polymerase Chain Reaction, Infection, Amplification, Derivative Assay

7) Product Images from "Identification of a Broadly Cross-Reactive Epitope in the Inner Shell of the Norovirus Capsid"

Article Title: Identification of a Broadly Cross-Reactive Epitope in the Inner Shell of the Norovirus Capsid

Journal: PLoS ONE

doi: 10.1371/journal.pone.0067592

Reactivity of MAb TV20 with NV capsid proteins carrying point mutations. Immunofluorescence staining results from Vero cells transfected with different DNA constructs. Mutations of the VP1 from NV (pCI-NV) were introduced by using specific primers and the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) following the manufacturer's recommendations. pCI expressing GFP was used as negative control. Hyperimmune sera against NV (α-NV) was used as a control to detect VP1 expression.
Figure Legend Snippet: Reactivity of MAb TV20 with NV capsid proteins carrying point mutations. Immunofluorescence staining results from Vero cells transfected with different DNA constructs. Mutations of the VP1 from NV (pCI-NV) were introduced by using specific primers and the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) following the manufacturer's recommendations. pCI expressing GFP was used as negative control. Hyperimmune sera against NV (α-NV) was used as a control to detect VP1 expression.

Techniques Used: Immunofluorescence, Staining, Transfection, Construct, Mutagenesis, Expressing, Negative Control

8) Product Images from "Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development"

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development

Journal: PLoS ONE

doi: 10.1371/journal.pone.0028537

Analysis of RAX mRNA expression in the brains of rep mice. Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.
Figure Legend Snippet: Analysis of RAX mRNA expression in the brains of rep mice. Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.

Techniques Used: Expressing, Mouse Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

9) Product Images from "Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry"

Article Title: Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry

Journal: Retrovirology

doi: 10.1186/1742-4690-6-3

MuPAR and HuPAR2 chimeras reveal regions required for PERV-A 14/220* infection . MuPAR is not permissive for PERV-A binding and entry, while HuPAR2 is permissive for both. Chimeras were constructed by swapping regions of HuPAR2 (solid black) with the corresponding residues of MuPAR (hatched black). Constructs were tagged with either an N-terminal c-myc tag (open circle) or a C-terminal eGFP tag (gray oval), as a way to monitor expression. Chimeras were expressed in non-permissive SIRC cells and tested for PERV-A infection. Levels of infection were determined by PERV pol qPCR of 250 ng genomic DNA and compared to wild-type HuPAR2 and MuPAR. (-/+) indicates the status of PERV-A infection. The average PERV pol copy numbers and standard deviations ( n = 3) are shown for each. These chimeras revealed that the N-terminal 135 amino acids are critical for PERV-A 14/220* infection.
Figure Legend Snippet: MuPAR and HuPAR2 chimeras reveal regions required for PERV-A 14/220* infection . MuPAR is not permissive for PERV-A binding and entry, while HuPAR2 is permissive for both. Chimeras were constructed by swapping regions of HuPAR2 (solid black) with the corresponding residues of MuPAR (hatched black). Constructs were tagged with either an N-terminal c-myc tag (open circle) or a C-terminal eGFP tag (gray oval), as a way to monitor expression. Chimeras were expressed in non-permissive SIRC cells and tested for PERV-A infection. Levels of infection were determined by PERV pol qPCR of 250 ng genomic DNA and compared to wild-type HuPAR2 and MuPAR. (-/+) indicates the status of PERV-A infection. The average PERV pol copy numbers and standard deviations ( n = 3) are shown for each. These chimeras revealed that the N-terminal 135 amino acids are critical for PERV-A 14/220* infection.

Techniques Used: Infection, Binding Assay, Construct, Expressing, Real-time Polymerase Chain Reaction

HuPAR1 and HuPAR2 function for PERV-A 14/220* infection . HuPAR1 and HuPAR2 C-terminally tagged eGFP constructs were expressed in non-permissive SIRC cells. Stable lines were sorted by eGFP expression to yield cell populations with similar receptor expression levels. PERV pol copy number in 250 ng genomic DNA of infected SIRC/receptor-expressing cell lines was determined to assess receptor function. HuPAR2 PERV pol copy number was normalized by HuPAR1 PERV pol copy number in each experiment and expressed as percent of HuPAR1 function. The average function determined by three individual infection experiments with three replicates each is shown with standard errors. HuPAR2 is 11-fold more functional than HuPAR1 (p
Figure Legend Snippet: HuPAR1 and HuPAR2 function for PERV-A 14/220* infection . HuPAR1 and HuPAR2 C-terminally tagged eGFP constructs were expressed in non-permissive SIRC cells. Stable lines were sorted by eGFP expression to yield cell populations with similar receptor expression levels. PERV pol copy number in 250 ng genomic DNA of infected SIRC/receptor-expressing cell lines was determined to assess receptor function. HuPAR2 PERV pol copy number was normalized by HuPAR1 PERV pol copy number in each experiment and expressed as percent of HuPAR1 function. The average function determined by three individual infection experiments with three replicates each is shown with standard errors. HuPAR2 is 11-fold more functional than HuPAR1 (p

Techniques Used: Infection, Construct, Expressing, Functional Assay

10) Product Images from "Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development"

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development

Journal: PLoS ONE

doi: 10.1371/journal.pone.0028537

Analysis of RAX mRNA expression in the brains of rep mice. Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.
Figure Legend Snippet: Analysis of RAX mRNA expression in the brains of rep mice. Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.

Techniques Used: Expressing, Mouse Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

11) Product Images from "Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development"

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development

Journal: PLoS ONE

doi: 10.1371/journal.pone.0028537

Analysis of RAX mRNA expression in the brains of rep mice. Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.
Figure Legend Snippet: Analysis of RAX mRNA expression in the brains of rep mice. Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.

Techniques Used: Expressing, Mouse Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

12) Product Images from "Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development"

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development

Journal: PLoS ONE

doi: 10.1371/journal.pone.0028537

Analysis of RAX mRNA expression in the brains of rep mice. Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.
Figure Legend Snippet: Analysis of RAX mRNA expression in the brains of rep mice. Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.

Techniques Used: Expressing, Mouse Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

13) Product Images from "Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection"

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx113

In vivo transposition in prokaryotic cells. ( A ) Scheme of the experimental method. The donor plasmid DNA, carrying a gene of interest (kanamycin resistance cassette, Kan R ) flanked with (IRs), contains a conditional origin of replication, oriR6K, which prevents replication in the recipient strain. Donor plasmid DNA and purified recombinant transposase (Mos1 or Mboumar-9) are co-transfected into bacterial cells, resulting in integration of the kanamycin cassette into the genomic DNA. ( B ) Kanamycin resistant colonies were obtained only if purified transposase was included in the transfection reaction. White colonies on dark background. ( C ) Relative efficiency of transposition observed after treatment of the reactions, prior to transfection, with proteinase K, phenol, no treatment and the control (no transposase added). Two technical repeats were performed for two biological repeats. ( D ) Agarose gel of the products of colony polymerase chain reaction (PCR) to detect the presence of the donor plasmid in the kanamycin resistant colonies. Clone 1 (Lane 4) has traces of the donor plasmid backbone detected. Positive control—a colony of Escherichia coli S17 λ pir carrying donor plasmid; negative control—a colony of the recipient strain E. coli DH10B. ( E ) Five analyzed clones are resistant to kanamycin, but sensitive to chloramphenicol—the plasmid backbone resistance. Controls as in (D). ( F ) Southern Blotting analysis of the digested genomic DNA from the kanamycin resistant clones hybridized with fluorescently labeled IR DNA. Negative control: genomic DNA of the recipient strain E. coli DH10B. ( G ) WebLogo alignment of 40 bp around the TA target nucleotides duplication of the 14 integration sites by Mos1 in the bacterial genome.
Figure Legend Snippet: In vivo transposition in prokaryotic cells. ( A ) Scheme of the experimental method. The donor plasmid DNA, carrying a gene of interest (kanamycin resistance cassette, Kan R ) flanked with (IRs), contains a conditional origin of replication, oriR6K, which prevents replication in the recipient strain. Donor plasmid DNA and purified recombinant transposase (Mos1 or Mboumar-9) are co-transfected into bacterial cells, resulting in integration of the kanamycin cassette into the genomic DNA. ( B ) Kanamycin resistant colonies were obtained only if purified transposase was included in the transfection reaction. White colonies on dark background. ( C ) Relative efficiency of transposition observed after treatment of the reactions, prior to transfection, with proteinase K, phenol, no treatment and the control (no transposase added). Two technical repeats were performed for two biological repeats. ( D ) Agarose gel of the products of colony polymerase chain reaction (PCR) to detect the presence of the donor plasmid in the kanamycin resistant colonies. Clone 1 (Lane 4) has traces of the donor plasmid backbone detected. Positive control—a colony of Escherichia coli S17 λ pir carrying donor plasmid; negative control—a colony of the recipient strain E. coli DH10B. ( E ) Five analyzed clones are resistant to kanamycin, but sensitive to chloramphenicol—the plasmid backbone resistance. Controls as in (D). ( F ) Southern Blotting analysis of the digested genomic DNA from the kanamycin resistant clones hybridized with fluorescently labeled IR DNA. Negative control: genomic DNA of the recipient strain E. coli DH10B. ( G ) WebLogo alignment of 40 bp around the TA target nucleotides duplication of the 14 integration sites by Mos1 in the bacterial genome.

Techniques Used: In Vivo, Plasmid Preparation, Purification, Recombinant, Transfection, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Positive Control, Negative Control, Clone Assay, Southern Blot, Labeling

14) Product Images from "Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development"

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development

Journal: PLoS ONE

doi: 10.1371/journal.pone.0028537

Analysis of RAX mRNA expression in the brains of rep mice. Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.
Figure Legend Snippet: Analysis of RAX mRNA expression in the brains of rep mice. Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.

Techniques Used: Expressing, Mouse Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

15) Product Images from "Nuclear factor I-A represses expression of the cell adhesion molecule L1"

Article Title: Nuclear factor I-A represses expression of the cell adhesion molecule L1

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-10-107

NFI-A binds to the L1 gene in vivo . A , ChIP analysis of N2A cells expressing Myc-NFI-A. L1 , NFI-A-DNA complexes in the region of the L1 gene containing the NFI full binding site were assayed by PCR using primers indicated in Fig. 1A. Lanes: NFI-A bs , precipitation from Myc-NFI-A bs-transfected cells; NFI-A st , precipitation from Myc-NFI-A st-transfected cells; mock , precipitation from mock-transfected cells; pos , positive control (mouse genomic DNA as PCR template); water , water control (without PCR template). Chst8 and Chst11 , To further confirm the specificity of ChIP analysis, ChIP samples were analyzed by PCR using primers amplifying parts of the mouse Chst8 and Chst11 genes, respectively. Note the absence of PCR signals for Chst8 and Chst11 in the ChIP sample lanes. B , Expression of Myc-tagged NFI-A isoforms in N2A cells. N2A cells were transfected with 4.8 μg of NFI-A cDNA expression vectors to express Myc-NFI-A st or Myc-NFI-A bs, respectively, or with 4.8 μg pCMX plasmid (
Figure Legend Snippet: NFI-A binds to the L1 gene in vivo . A , ChIP analysis of N2A cells expressing Myc-NFI-A. L1 , NFI-A-DNA complexes in the region of the L1 gene containing the NFI full binding site were assayed by PCR using primers indicated in Fig. 1A. Lanes: NFI-A bs , precipitation from Myc-NFI-A bs-transfected cells; NFI-A st , precipitation from Myc-NFI-A st-transfected cells; mock , precipitation from mock-transfected cells; pos , positive control (mouse genomic DNA as PCR template); water , water control (without PCR template). Chst8 and Chst11 , To further confirm the specificity of ChIP analysis, ChIP samples were analyzed by PCR using primers amplifying parts of the mouse Chst8 and Chst11 genes, respectively. Note the absence of PCR signals for Chst8 and Chst11 in the ChIP sample lanes. B , Expression of Myc-tagged NFI-A isoforms in N2A cells. N2A cells were transfected with 4.8 μg of NFI-A cDNA expression vectors to express Myc-NFI-A st or Myc-NFI-A bs, respectively, or with 4.8 μg pCMX plasmid ("mock"). 48 h after transfection, cell were lysed and whole cell extracts were analyzed on a 10% SDS-PAGE gel, transferred to nitrocellulose membrane, and probed with anti-Myc antibody for detection of recombinant Myc-NFI-A and with anti-GAPDH antibody to check for loading of comparable protein amounts.

Techniques Used: In Vivo, Chromatin Immunoprecipitation, Expressing, Binding Assay, Polymerase Chain Reaction, Transfection, Positive Control, Plasmid Preparation, SDS Page, Recombinant

NFI-A binds to its full binding site in the mouse L1 gene in vitro . 32 P-labeled oligonucleotides used for EMSA: L: full NFI binding site in mouse L1 gene with flanking sequences; Lc: point-mutated variant of L; N: idealized NFI binding site with flanking sequences [ 18 ]; Nc: point-mutated variant of N; C: oligonucleotide without NFI recognition motif (SIS oligonucleotide). NFI binding site sequences are capitalized , point mutations are shown in boldface . The sequence of one strand is shown after the fill-in reaction. Oligonucleotides were incubated with extracts from CHO cells expressing NFI-A bs or NFI-A st, or from mock-transfected cells (CMV). Only the upper and lower parts of the autoradiogram are shown, the middle section, which did not exhibit any signals, was cut out for reasons of space. Arrows A: HA-NFI-A-DNA complexes; arrow B: complexes of endogenous NFI and DNA; arrows C and D: free oligonucleotide DNA. Note that that the free SIS oligonucleotide migrates slightly slower ( arrow C) than the other free oligonucleotides ( arrow D) due to its higher molecular weight.
Figure Legend Snippet: NFI-A binds to its full binding site in the mouse L1 gene in vitro . 32 P-labeled oligonucleotides used for EMSA: L: full NFI binding site in mouse L1 gene with flanking sequences; Lc: point-mutated variant of L; N: idealized NFI binding site with flanking sequences [ 18 ]; Nc: point-mutated variant of N; C: oligonucleotide without NFI recognition motif (SIS oligonucleotide). NFI binding site sequences are capitalized , point mutations are shown in boldface . The sequence of one strand is shown after the fill-in reaction. Oligonucleotides were incubated with extracts from CHO cells expressing NFI-A bs or NFI-A st, or from mock-transfected cells (CMV). Only the upper and lower parts of the autoradiogram are shown, the middle section, which did not exhibit any signals, was cut out for reasons of space. Arrows A: HA-NFI-A-DNA complexes; arrow B: complexes of endogenous NFI and DNA; arrows C and D: free oligonucleotide DNA. Note that that the free SIS oligonucleotide migrates slightly slower ( arrow C) than the other free oligonucleotides ( arrow D) due to its higher molecular weight.

Techniques Used: Binding Assay, In Vitro, Labeling, Variant Assay, Sequencing, Incubation, Expressing, Transfection, Molecular Weight

In vitro binding of NFI-A to its full binding site in the mouse L1 gene is confirmed by supershift and competition assays . EMSAs were performed with 32 P-labeled oligonucleotides indicated above the autoradiograms (for a description, see Fig. 3). In both images ( A-B ), only the upper and lower parts of the respective autoradiogram are shown, the middle sections, which did not exhibit any signals, were cut out for reasons of space. A , Supershift experiment. NFI-A st
Figure Legend Snippet: In vitro binding of NFI-A to its full binding site in the mouse L1 gene is confirmed by supershift and competition assays . EMSAs were performed with 32 P-labeled oligonucleotides indicated above the autoradiograms (for a description, see Fig. 3). In both images ( A-B ), only the upper and lower parts of the respective autoradiogram are shown, the middle sections, which did not exhibit any signals, were cut out for reasons of space. A , Supershift experiment. NFI-A st "+": oligonucleotides were incubated with extracts from CHO cells transfected with pCHNFI-A (NFI-A st); NFI-A st "-": oligonucleotides were not pre-incubated with cell extracts (negative control). Where indicated, anti-HA antibody was added. Arrow a points at anti-HA-antibody/NFI-A/DNA super-shifted complexes, while arrow b indicates NFI-A/DNA complexes. B , Competition analysis with the radioactively labeled NFI binding site from the mouse L1 gene regulatory region (L) and various unlabeled competitor oligonucleotides (N, Nc, L, Lc; see Fig. 3 for a description). The 32 P-labeled L oligonucleotide was incubated with extracts from CHO cells transfected with pCHNFI-A (NFI-A st) in the presence of different molar excesses of the unlabeled competitor oligonucleotides as indicated above the autoradiogram. A "-" indicates that oligonucleotides were not pre-incubated with cell extracts (negative control). The arrow points at the main band representing NFI-A-DNA complexes.

Techniques Used: In Vitro, Binding Assay, Labeling, Incubation, Transfection, Negative Control

NFI-A represses mouse L1 gene expression . Results of luciferase-based reporter gene assays in N2A cells transfected with L1-11. The L1 reporter plasmid L1-11 contains 2943 bp upstream of exon 1 of the mouse L1 gene, exon 1, intron 1, exon 2 with the luciferase cDNA inserted to replace the L1 start codon by the luciferase start codon, intron 2 including the neural restrictive silencer element, exon 3, intron 3 and exon 4. beta Gal: coexpression of beta-galactosidase (negative control) ( n = 98); NFI-A bs: coexpression of NFI-A bs ( n = 91); NFI-A bs del: coexpression of NFI-A bs del ( n = 35). For titration of beta Gal with NFI-A bs, the beta Gal expression plasmid was gradually replaced with the NFI-A bs expression plasmid at the ratios indicated below, leaving the total amount of transfected DNA constant.
Figure Legend Snippet: NFI-A represses mouse L1 gene expression . Results of luciferase-based reporter gene assays in N2A cells transfected with L1-11. The L1 reporter plasmid L1-11 contains 2943 bp upstream of exon 1 of the mouse L1 gene, exon 1, intron 1, exon 2 with the luciferase cDNA inserted to replace the L1 start codon by the luciferase start codon, intron 2 including the neural restrictive silencer element, exon 3, intron 3 and exon 4. beta Gal: coexpression of beta-galactosidase (negative control) ( n = 98); NFI-A bs: coexpression of NFI-A bs ( n = 91); NFI-A bs del: coexpression of NFI-A bs del ( n = 35). For titration of beta Gal with NFI-A bs, the beta Gal expression plasmid was gradually replaced with the NFI-A bs expression plasmid at the ratios indicated below, leaving the total amount of transfected DNA constant. "1", bs:Gal = 1:59 ( n = 28); "2", bs:Gal = 1:11 ( n = 35); "3", bs:Gal = 1:5 ( n = 49); "4", bs:Gal = 1:2 ( n = 21); "5", bs:Gal = 1:1 ( n = 49); "6", bs:Gal = 2:1 ( n = 21). Data were acquired from 3 (bs:Gal = 1:2) to 9 (beta Gal, NFI-A bs) independent experiments. For normalization, luciferase activity was divided by the fluorescence of coexpressed EGFP. The relative luciferase activity of the beta Gal-transfected samples was set to 1. **, p

Techniques Used: Expressing, Luciferase, Transfection, Plasmid Preparation, Negative Control, Titration, Activity Assay, Fluorescence

16) Product Images from "Development of a Rapid PCR Method Using the Insertion Sequence IS1203 for Genotyping Shiga Toxin-Producing Escherichia coli O157"

Article Title: Development of a Rapid PCR Method Using the Insertion Sequence IS1203 for Genotyping Shiga Toxin-Producing Escherichia coli O157

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.42.12.5462-5466.2004

Schematic diagram of IS 1203 PCR typing. (a) IS 1203 sequences were divided into two fragments, the upstream (open box) and downstream (solid box) fragments, by PvuII digestion between nucleotides 1032 and 1033 (arrowhead). Arrows indicate the primer regions for upstream typing (1203-82 and 1203-11 for first PCR, and 1203-72 and 1203-21 for nested PCR) and downstream typing (1203-62 and 1203-31 for first PCR, and 1203-52 and 1203-41 for nested PCR). (b) Detection of PvuII-digested fragments containing IS 1203 . STEC O157 genomic DNA contains more than 10 copies of IS 1203 (two copies of IS 1203 are shown in the figure for simplicity). Open box, IS1203 upstream region; solid box, downstream region. STEC O157 genomic DNA was digested at the PvuII recognition sites (arrowheads). Digested fragments were then self-ligated. Ligated fragments containing IS 1203 were amplified by PCR in the direction of the arrows. Fragments containing upstream or downstream regions of IS 1203 were reacted. Nested PCR was performed for accuracy, and two electrophoresis patterns (upstream and downstream patterns) were obtained from one strain.
Figure Legend Snippet: Schematic diagram of IS 1203 PCR typing. (a) IS 1203 sequences were divided into two fragments, the upstream (open box) and downstream (solid box) fragments, by PvuII digestion between nucleotides 1032 and 1033 (arrowhead). Arrows indicate the primer regions for upstream typing (1203-82 and 1203-11 for first PCR, and 1203-72 and 1203-21 for nested PCR) and downstream typing (1203-62 and 1203-31 for first PCR, and 1203-52 and 1203-41 for nested PCR). (b) Detection of PvuII-digested fragments containing IS 1203 . STEC O157 genomic DNA contains more than 10 copies of IS 1203 (two copies of IS 1203 are shown in the figure for simplicity). Open box, IS1203 upstream region; solid box, downstream region. STEC O157 genomic DNA was digested at the PvuII recognition sites (arrowheads). Digested fragments were then self-ligated. Ligated fragments containing IS 1203 were amplified by PCR in the direction of the arrows. Fragments containing upstream or downstream regions of IS 1203 were reacted. Nested PCR was performed for accuracy, and two electrophoresis patterns (upstream and downstream patterns) were obtained from one strain.

Techniques Used: Polymerase Chain Reaction, Nested PCR, Amplification, Electrophoresis

Related Articles

Countercurrent Chromatography:

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development
Article Snippet: .. The resulting overlapping PCR products were then used as template for a second round PCR using the primers: 5′ AAC TCG AGG TAC CAT GGA CTA CTA CAA GG and 3′ AAT CTA GAG GAT CCC TAC TTT CTT TCT GCT ATT ATC TTT AAA T with Expand High Fidelity PCR System (Roche), the mutagenized product was digested with XhoI and XbaI (New England Biolabs) and ligated into pLVX-IRES-ZsGreen1 (Clontech) with Rapid DNA Ligation Kit (Roche) to generate pLVX-FLAG-RAX(S130P)-IRES-ZsGreen1. .. Construction of the RAX shRNA expression clone The DNA oligonucleotides CCG GCC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG TTT TTG and AAT TCA AAA ACC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG were annealed and ligated into pLKO.1-puro digested with AgeI and EcoRI (New England Biolabs) using Rapid DNA Ligation Kit (Roche) to generate pLKO.1-RAX (1199)-puro.

Clone Assay:

Article Title: Identification of a Broadly Cross-Reactive Epitope in the Inner Shell of the Norovirus Capsid
Article Snippet: .. A pCI-GFP was used as a template and amplicons were digested with XbaI and SalI and cloned into a pCI vector with Rapid DNA Ligation Kit (Roche Applied Science, Germany). .. Each of the products was transformed into TOP10 competent cells (Invitrogen).

Article Title: Frequency of Spontaneous Mutations in an Avian Hepadnavirus Infection
Article Snippet: .. For cloning, PCR amplification products from serum were digested with the restriction enzymes Apa I and Kpn I (both from New England BioLabs) and ligated into an appropriately cut pGEM-7Zf(+) vector (Promega) by using a rapid DNA ligation kit (Roche Molecular Biochemicals, Indianapolis, Ind.). .. Recombinant plasmids were cloned into DH5α bacterial cells (GIBCO/BRL, Gaithersburg, Md.).

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: .. The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. DNA primers were synthesized by Integrated Technologies, Inc. Tev protease was obtained from Invitrogen.

shRNA:

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development
Article Snippet: .. Construction of the RAX shRNA expression clone The DNA oligonucleotides CCG GCC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG TTT TTG and AAT TCA AAA ACC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG were annealed and ligated into pLKO.1-puro digested with AgeI and EcoRI (New England Biolabs) using Rapid DNA Ligation Kit (Roche) to generate pLKO.1-RAX (1199)-puro. .. Production of and infection with recombinant lentiviruses The lentiviral plasmids pLVX-IRES-ZsGreen1, pLVX-FLAG-RAX-IRES-ZsGreen1, pLVX-FLAG-RAX (S130P)-IRES-ZsGreen1 or pLKO.1-RAX (1199)-puro were cotransfected with the packaging plasmid pCMV-dR8.74 and the pseudotyping plasmid pVSV-G by calcium phosphate into HEK293T cells.

Agarose Gel Electrophoresis:

Article Title: A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification
Article Snippet: .. The DNA was isolated from the agarose gel slice by using GeneClean II (Bio 101 Systems/Qbiogene) and ligated into dephosphorylated BamHI-cut pUC18, using the Rapid DNA ligation kit (Roche Diagnostics Belgium). .. After transformation of One Shot MAX Efficiency DH5α-T1 Escherichia coli (Invitrogen), the bacteria were incubated for blue-white colony screening on agar plates containing X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside).

DNA Ligation:

Article Title: Overexpression of VEGF165b in Podocytes Reduces Glomerular Permeability
Article Snippet: .. The nephrin promoter (courtesy of Prof. S Quaggin) DNA fragment was inserted with rapid DNA ligation kit (Roche Applied Science) into the linearized pcDNA3-VEGF165 b, from which the cytomegalovirus promoter had been deleted and correctly oriented colonies selected. .. Human conditionally immortalized visceral glomerular epithelial cells previously characterized (donated by Prof. Moin Saleem) were transfected with equal amounts of pNephrin-VEGF165 b and empty vector 5′-Nephrin-pKO.

Article Title: Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load
Article Snippet: .. These PCR products were ligated by a Rapid DNA ligation kit (Roche Applied Science, Penzberg, Germany) after EcoRI digestion. .. The ligated PCR product was then further ligated into pCR-Blunt II-TOPO using a Zero Blunt TOPO PCR cloning kit (Life Technologies), according to the manufacturer's protocol.

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development
Article Snippet: .. Construction of the RAX shRNA expression clone The DNA oligonucleotides CCG GCC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG TTT TTG and AAT TCA AAA ACC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG were annealed and ligated into pLKO.1-puro digested with AgeI and EcoRI (New England Biolabs) using Rapid DNA Ligation Kit (Roche) to generate pLKO.1-RAX (1199)-puro. .. Production of and infection with recombinant lentiviruses The lentiviral plasmids pLVX-IRES-ZsGreen1, pLVX-FLAG-RAX-IRES-ZsGreen1, pLVX-FLAG-RAX (S130P)-IRES-ZsGreen1 or pLKO.1-RAX (1199)-puro were cotransfected with the packaging plasmid pCMV-dR8.74 and the pseudotyping plasmid pVSV-G by calcium phosphate into HEK293T cells.

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development
Article Snippet: .. The resulting overlapping PCR products were then used as template for a second round PCR using the primers: 5′ AAC TCG AGG TAC CAT GGA CTA CTA CAA GG and 3′ AAT CTA GAG GAT CCC TAC TTT CTT TCT GCT ATT ATC TTT AAA T with Expand High Fidelity PCR System (Roche), the mutagenized product was digested with XhoI and XbaI (New England Biolabs) and ligated into pLVX-IRES-ZsGreen1 (Clontech) with Rapid DNA Ligation Kit (Roche) to generate pLVX-FLAG-RAX(S130P)-IRES-ZsGreen1. .. Construction of the RAX shRNA expression clone The DNA oligonucleotides CCG GCC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG TTT TTG and AAT TCA AAA ACC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG were annealed and ligated into pLKO.1-puro digested with AgeI and EcoRI (New England Biolabs) using Rapid DNA Ligation Kit (Roche) to generate pLKO.1-RAX (1199)-puro.

Article Title: Identification of a Broadly Cross-Reactive Epitope in the Inner Shell of the Norovirus Capsid
Article Snippet: .. A pCI-GFP was used as a template and amplicons were digested with XbaI and SalI and cloned into a pCI vector with Rapid DNA Ligation Kit (Roche Applied Science, Germany). .. Each of the products was transformed into TOP10 competent cells (Invitrogen).

Article Title: A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification
Article Snippet: .. The DNA was isolated from the agarose gel slice by using GeneClean II (Bio 101 Systems/Qbiogene) and ligated into dephosphorylated BamHI-cut pUC18, using the Rapid DNA ligation kit (Roche Diagnostics Belgium). .. After transformation of One Shot MAX Efficiency DH5α-T1 Escherichia coli (Invitrogen), the bacteria were incubated for blue-white colony screening on agar plates containing X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside).

Article Title: Frequency of Spontaneous Mutations in an Avian Hepadnavirus Infection
Article Snippet: .. For cloning, PCR amplification products from serum were digested with the restriction enzymes Apa I and Kpn I (both from New England BioLabs) and ligated into an appropriately cut pGEM-7Zf(+) vector (Promega) by using a rapid DNA ligation kit (Roche Molecular Biochemicals, Indianapolis, Ind.). .. Recombinant plasmids were cloned into DH5α bacterial cells (GIBCO/BRL, Gaithersburg, Md.).

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: .. The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. DNA primers were synthesized by Integrated Technologies, Inc. Tev protease was obtained from Invitrogen.

Polymerase Chain Reaction:

Article Title: Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load
Article Snippet: .. These PCR products were ligated by a Rapid DNA ligation kit (Roche Applied Science, Penzberg, Germany) after EcoRI digestion. .. The ligated PCR product was then further ligated into pCR-Blunt II-TOPO using a Zero Blunt TOPO PCR cloning kit (Life Technologies), according to the manufacturer's protocol.

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development
Article Snippet: .. The resulting overlapping PCR products were then used as template for a second round PCR using the primers: 5′ AAC TCG AGG TAC CAT GGA CTA CTA CAA GG and 3′ AAT CTA GAG GAT CCC TAC TTT CTT TCT GCT ATT ATC TTT AAA T with Expand High Fidelity PCR System (Roche), the mutagenized product was digested with XhoI and XbaI (New England Biolabs) and ligated into pLVX-IRES-ZsGreen1 (Clontech) with Rapid DNA Ligation Kit (Roche) to generate pLVX-FLAG-RAX(S130P)-IRES-ZsGreen1. .. Construction of the RAX shRNA expression clone The DNA oligonucleotides CCG GCC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG TTT TTG and AAT TCA AAA ACC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG were annealed and ligated into pLKO.1-puro digested with AgeI and EcoRI (New England Biolabs) using Rapid DNA Ligation Kit (Roche) to generate pLKO.1-RAX (1199)-puro.

Article Title: Frequency of Spontaneous Mutations in an Avian Hepadnavirus Infection
Article Snippet: .. For cloning, PCR amplification products from serum were digested with the restriction enzymes Apa I and Kpn I (both from New England BioLabs) and ligated into an appropriately cut pGEM-7Zf(+) vector (Promega) by using a rapid DNA ligation kit (Roche Molecular Biochemicals, Indianapolis, Ind.). .. Recombinant plasmids were cloned into DH5α bacterial cells (GIBCO/BRL, Gaithersburg, Md.).

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: .. The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. DNA primers were synthesized by Integrated Technologies, Inc. Tev protease was obtained from Invitrogen.

Isolation:

Article Title: A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification
Article Snippet: .. The DNA was isolated from the agarose gel slice by using GeneClean II (Bio 101 Systems/Qbiogene) and ligated into dephosphorylated BamHI-cut pUC18, using the Rapid DNA ligation kit (Roche Diagnostics Belgium). .. After transformation of One Shot MAX Efficiency DH5α-T1 Escherichia coli (Invitrogen), the bacteria were incubated for blue-white colony screening on agar plates containing X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside).

Construct:

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: .. The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. DNA primers were synthesized by Integrated Technologies, Inc. Tev protease was obtained from Invitrogen.

Purification:

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: .. The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. DNA primers were synthesized by Integrated Technologies, Inc. Tev protease was obtained from Invitrogen.

CTG Assay:

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development
Article Snippet: .. Construction of the RAX shRNA expression clone The DNA oligonucleotides CCG GCC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG TTT TTG and AAT TCA AAA ACC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG were annealed and ligated into pLKO.1-puro digested with AgeI and EcoRI (New England Biolabs) using Rapid DNA Ligation Kit (Roche) to generate pLKO.1-RAX (1199)-puro. .. Production of and infection with recombinant lentiviruses The lentiviral plasmids pLVX-IRES-ZsGreen1, pLVX-FLAG-RAX-IRES-ZsGreen1, pLVX-FLAG-RAX (S130P)-IRES-ZsGreen1 or pLKO.1-RAX (1199)-puro were cotransfected with the packaging plasmid pCMV-dR8.74 and the pseudotyping plasmid pVSV-G by calcium phosphate into HEK293T cells.

Amplification:

Article Title: Frequency of Spontaneous Mutations in an Avian Hepadnavirus Infection
Article Snippet: .. For cloning, PCR amplification products from serum were digested with the restriction enzymes Apa I and Kpn I (both from New England BioLabs) and ligated into an appropriately cut pGEM-7Zf(+) vector (Promega) by using a rapid DNA ligation kit (Roche Molecular Biochemicals, Indianapolis, Ind.). .. Recombinant plasmids were cloned into DH5α bacterial cells (GIBCO/BRL, Gaithersburg, Md.).

Expressing:

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development
Article Snippet: .. Construction of the RAX shRNA expression clone The DNA oligonucleotides CCG GCC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG TTT TTG and AAT TCA AAA ACC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG were annealed and ligated into pLKO.1-puro digested with AgeI and EcoRI (New England Biolabs) using Rapid DNA Ligation Kit (Roche) to generate pLKO.1-RAX (1199)-puro. .. Production of and infection with recombinant lentiviruses The lentiviral plasmids pLVX-IRES-ZsGreen1, pLVX-FLAG-RAX-IRES-ZsGreen1, pLVX-FLAG-RAX (S130P)-IRES-ZsGreen1 or pLKO.1-RAX (1199)-puro were cotransfected with the packaging plasmid pCMV-dR8.74 and the pseudotyping plasmid pVSV-G by calcium phosphate into HEK293T cells.

Sequencing:

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: .. The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. DNA primers were synthesized by Integrated Technologies, Inc. Tev protease was obtained from Invitrogen.

Chloramphenicol Acetyltransferase Assay:

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development
Article Snippet: .. The resulting overlapping PCR products were then used as template for a second round PCR using the primers: 5′ AAC TCG AGG TAC CAT GGA CTA CTA CAA GG and 3′ AAT CTA GAG GAT CCC TAC TTT CTT TCT GCT ATT ATC TTT AAA T with Expand High Fidelity PCR System (Roche), the mutagenized product was digested with XhoI and XbaI (New England Biolabs) and ligated into pLVX-IRES-ZsGreen1 (Clontech) with Rapid DNA Ligation Kit (Roche) to generate pLVX-FLAG-RAX(S130P)-IRES-ZsGreen1. .. Construction of the RAX shRNA expression clone The DNA oligonucleotides CCG GCC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG TTT TTG and AAT TCA AAA ACC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG were annealed and ligated into pLKO.1-puro digested with AgeI and EcoRI (New England Biolabs) using Rapid DNA Ligation Kit (Roche) to generate pLKO.1-RAX (1199)-puro.

Cellular Antioxidant Activity Assay:

Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development
Article Snippet: .. The resulting overlapping PCR products were then used as template for a second round PCR using the primers: 5′ AAC TCG AGG TAC CAT GGA CTA CTA CAA GG and 3′ AAT CTA GAG GAT CCC TAC TTT CTT TCT GCT ATT ATC TTT AAA T with Expand High Fidelity PCR System (Roche), the mutagenized product was digested with XhoI and XbaI (New England Biolabs) and ligated into pLVX-IRES-ZsGreen1 (Clontech) with Rapid DNA Ligation Kit (Roche) to generate pLVX-FLAG-RAX(S130P)-IRES-ZsGreen1. .. Construction of the RAX shRNA expression clone The DNA oligonucleotides CCG GCC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG TTT TTG and AAT TCA AAA ACC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG were annealed and ligated into pLKO.1-puro digested with AgeI and EcoRI (New England Biolabs) using Rapid DNA Ligation Kit (Roche) to generate pLKO.1-RAX (1199)-puro.

Plasmid Preparation:

Article Title: Identification of a Broadly Cross-Reactive Epitope in the Inner Shell of the Norovirus Capsid
Article Snippet: .. A pCI-GFP was used as a template and amplicons were digested with XbaI and SalI and cloned into a pCI vector with Rapid DNA Ligation Kit (Roche Applied Science, Germany). .. Each of the products was transformed into TOP10 competent cells (Invitrogen).

Article Title: Frequency of Spontaneous Mutations in an Avian Hepadnavirus Infection
Article Snippet: .. For cloning, PCR amplification products from serum were digested with the restriction enzymes Apa I and Kpn I (both from New England BioLabs) and ligated into an appropriately cut pGEM-7Zf(+) vector (Promega) by using a rapid DNA ligation kit (Roche Molecular Biochemicals, Indianapolis, Ind.). .. Recombinant plasmids were cloned into DH5α bacterial cells (GIBCO/BRL, Gaithersburg, Md.).

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: .. The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. DNA primers were synthesized by Integrated Technologies, Inc. Tev protease was obtained from Invitrogen.

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    Roche rapid dna ligation kit
    Schematic representation of the expression constructs ( A ) pLgals1-hum-β3GN-T2 and ( B ) pLgals1-hum-MFng . The <t>PCR-amplified</t> <t>DNA</t> sequences were inserted in the pLgals1-Tev vector construct at the multicloning site, which is preceded by the 6HisTag
    Rapid Dna Ligation Kit, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 188 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Schematic representation of the expression constructs ( A ) pLgals1-hum-β3GN-T2 and ( B ) pLgals1-hum-MFng . The PCR-amplified DNA sequences were inserted in the pLgals1-Tev vector construct at the multicloning site, which is preceded by the 6HisTag

    Journal: Glycobiology

    Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2

    doi: 10.1093/glycob/cwr110

    Figure Lengend Snippet: Schematic representation of the expression constructs ( A ) pLgals1-hum-β3GN-T2 and ( B ) pLgals1-hum-MFng . The PCR-amplified DNA sequences were inserted in the pLgals1-Tev vector construct at the multicloning site, which is preceded by the 6HisTag

    Article Snippet: The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem.

    Techniques: Expressing, Construct, Polymerase Chain Reaction, Amplification, Plasmid Preparation

    Analysis of RAX mRNA expression in the brains of rep mice. Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.

    Journal: PLoS ONE

    Article Title: Missense Mutation in the Second RNA Binding Domain Reveals a Role for Prkra (PACT/RAX) during Skull Development

    doi: 10.1371/journal.pone.0028537

    Figure Lengend Snippet: Analysis of RAX mRNA expression in the brains of rep mice. Total RNA was isolated from one WT and two rep mouse brains, treated with DNase and analyzed by RT-PCR with appropriate primers to measure the levels of the 5′ region (exons 1 and 2) and the 3′ region (exons 7 and 8) of RAX mRNA. 18S rRNA was measured as a loading control and –RT controls were used to ensure the absence of any genomic DNA in the RNA preparations.

    Article Snippet: Construction of the RAX shRNA expression clone The DNA oligonucleotides CCG GCC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG TTT TTG and AAT TCA AAA ACC GTC AAC TTT CCA GAT TTC TCG AGA AAT CTG GAA AGT TGA CGG were annealed and ligated into pLKO.1-puro digested with AgeI and EcoRI (New England Biolabs) using Rapid DNA Ligation Kit (Roche) to generate pLKO.1-RAX (1199)-puro.

    Techniques: Expressing, Mouse Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

    Generation of pNeph-VEGF 165 b heterozygous transgenic mice. (A) VEGF 165 b is cloned into an expression vector under the control of the Nephrin promoter (Transgenic Construct; Ai). When transfected into human podocytes, VEGF 165 b expression was significantly greater than control vector or untransfected cells (Aii). (B) PCR screen of pups born from injected embryos. (C) Southern blot, confirming single insertion into genomic DNA. The gDNA is digested with EcoRI, and a probe made from the cDNA is used to generate the transgene. There was a single EcoRI site in the 5′ end of the insert, so multiple insertions should generate multiple different-sized bands in the transgene. There is only a single band in line 1, indicating a single insertion point (insertions into multiple sites would generate multiple bands). See Supplemental Figure S1. Each lane in B and C represents a distinct animal line. The mouse nephrin sequence is also detected as seen in the WT animals (two bands as shown by arrows). Line 1 is used for establishment of the subsequent heterozygous and homozygous breeding colonies. (D) Of note, the ultrafiltration fraction from potential founder lines is not different from each other.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Overexpression of VEGF165b in Podocytes Reduces Glomerular Permeability

    doi: 10.1681/ASN.2009060617

    Figure Lengend Snippet: Generation of pNeph-VEGF 165 b heterozygous transgenic mice. (A) VEGF 165 b is cloned into an expression vector under the control of the Nephrin promoter (Transgenic Construct; Ai). When transfected into human podocytes, VEGF 165 b expression was significantly greater than control vector or untransfected cells (Aii). (B) PCR screen of pups born from injected embryos. (C) Southern blot, confirming single insertion into genomic DNA. The gDNA is digested with EcoRI, and a probe made from the cDNA is used to generate the transgene. There was a single EcoRI site in the 5′ end of the insert, so multiple insertions should generate multiple different-sized bands in the transgene. There is only a single band in line 1, indicating a single insertion point (insertions into multiple sites would generate multiple bands). See Supplemental Figure S1. Each lane in B and C represents a distinct animal line. The mouse nephrin sequence is also detected as seen in the WT animals (two bands as shown by arrows). Line 1 is used for establishment of the subsequent heterozygous and homozygous breeding colonies. (D) Of note, the ultrafiltration fraction from potential founder lines is not different from each other.

    Article Snippet: The nephrin promoter (courtesy of Prof. S Quaggin) DNA fragment was inserted with rapid DNA ligation kit (Roche Applied Science) into the linearized pcDNA3-VEGF165 b, from which the cytomegalovirus promoter had been deleted and correctly oriented colonies selected.

    Techniques: Transgenic Assay, Mouse Assay, Clone Assay, Expressing, Plasmid Preparation, Construct, Transfection, Polymerase Chain Reaction, Injection, Southern Blot, Sequencing

    Restriction enzyme analysis of the bovine tissue DNA before and after multiply primed RCA. One microliter of the nonamplified DNA, containing 3.9 μg of total DNA, was digested with EcoRI (lane 1). Multiply primed RCA with 450 μM extra dNTPs was performed with 1 μl (3.9 μg) of input bovine tissue DNA, and 2 μl of the RCA product was digested with EcoRI (lane 2), BamHI (lane 3), HindIII (lane 4), SalI (lane 5), and HincII (lane 6). As a negative control, 2 μl of multiply primed RCA product of water was digested with EcoRI (lane 7). Lane M, DNA molecular size marker (Fermentas).

    Journal: Journal of Virology

    Article Title: A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

    doi: 10.1128/JVI.78.10.4993-4998.2004

    Figure Lengend Snippet: Restriction enzyme analysis of the bovine tissue DNA before and after multiply primed RCA. One microliter of the nonamplified DNA, containing 3.9 μg of total DNA, was digested with EcoRI (lane 1). Multiply primed RCA with 450 μM extra dNTPs was performed with 1 μl (3.9 μg) of input bovine tissue DNA, and 2 μl of the RCA product was digested with EcoRI (lane 2), BamHI (lane 3), HindIII (lane 4), SalI (lane 5), and HincII (lane 6). As a negative control, 2 μl of multiply primed RCA product of water was digested with EcoRI (lane 7). Lane M, DNA molecular size marker (Fermentas).

    Article Snippet: The DNA was isolated from the agarose gel slice by using GeneClean II (Bio 101 Systems/Qbiogene) and ligated into dephosphorylated BamHI-cut pUC18, using the Rapid DNA ligation kit (Roche Diagnostics Belgium).

    Techniques: Negative Control, Marker