r0189  (New England Biolabs)


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    Name:
    NotI
    Description:
    NotI 2 500 units
    Catalog Number:
    R0189L
    Price:
    302
    Category:
    Restriction Enzymes
    Size:
    2 500 units
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    Structured Review

    New England Biolabs r0189
    NotI
    NotI 2 500 units
    https://www.bioz.com/result/r0189/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    r0189 - by Bioz Stars, 2021-06
    86/100 stars

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    Related Articles

    Pulsed-Field Gel:

    Article Title: Transmission of Yersinia pseudotuberculosis in the Pork Production Chain from Farm to Slaughterhouse ▿
    Article Snippet: Isolates were serotyped with slide agglutination using commercial O:1 to O:6 antisera (Denka Seiken, Tokyo, Japan). .. A total of 286 isolates were characterized by pulsed-field gel electrophoresis (PFGE) as described by Niskanen et al. ( ) using SpeI and NotI restriction enzymes (New England Biolabs, Ipswich, MA). ..

    Generated:

    Article Title: ATP signaling in the integrative neural center of Aplysia californica
    Article Snippet: The two isoforms of AcP2X vary by a 147 base deletion/insertion and would be difficult to distinguishable by in situ. .. The antisense probe was generated by digestion of the AcP2X plasmid with Not I (CatR0189s, New England Biolabs Inc.) then transcribed with T3 polymerase from the DIG RNA Labeling Kit (Cat#11175025910, Roche Diagnostics). .. The control sense probe was produced by the same protocol but used Pme1 (Cat# R0560s, New England Biolabs Inc.) for digestion and T7 polymerase for transcription.

    Article Title: ATP signaling in the integrative neural center of Aplysia californica
    Article Snippet: The two isoforms of Ac P2X vary by a 147 base deletion/insertion and would not be distinguishable by in situ . .. The antisense probe was generated by digestion of the AcP2X plasmid with Not I (CatR0189s, New England Biolabs Inc.) then transcribed with T3 polymerase from the DIG RNA Labeling Kit (Cat#11175025910, Roche Diagnostics). .. The control sense probe was produced by the same protocol but used Pme1 (Cat# R0560s, New England Biolabs Inc.) for digestion and T7 polymerase for transcription.

    Plasmid Preparation:

    Article Title: ATP signaling in the integrative neural center of Aplysia californica
    Article Snippet: The two isoforms of AcP2X vary by a 147 base deletion/insertion and would be difficult to distinguishable by in situ. .. The antisense probe was generated by digestion of the AcP2X plasmid with Not I (CatR0189s, New England Biolabs Inc.) then transcribed with T3 polymerase from the DIG RNA Labeling Kit (Cat#11175025910, Roche Diagnostics). .. The control sense probe was produced by the same protocol but used Pme1 (Cat# R0560s, New England Biolabs Inc.) for digestion and T7 polymerase for transcription.

    Article Title: ATP signaling in the integrative neural center of Aplysia californica
    Article Snippet: The two isoforms of Ac P2X vary by a 147 base deletion/insertion and would not be distinguishable by in situ . .. The antisense probe was generated by digestion of the AcP2X plasmid with Not I (CatR0189s, New England Biolabs Inc.) then transcribed with T3 polymerase from the DIG RNA Labeling Kit (Cat#11175025910, Roche Diagnostics). .. The control sense probe was produced by the same protocol but used Pme1 (Cat# R0560s, New England Biolabs Inc.) for digestion and T7 polymerase for transcription.

    Article Title: N-glycosylation of Viral E Protein Is the Determinant for Vector Midgut Invasion by Flaviviruses
    Article Snippet: .. All of the segments were further assembled into low-copy-number vector PACYC177 with a T7 promoter in front of the 5′ terminus and a NotI restriction site following the 3′ end using NEBuilder Hi-Fi DNA assembly master mix (NEB Biolabs). .. The Natal-RGN strain was synthesized using sequence as the template and divided into three pieces, including A (positions 1 to 3370), B (positions 3325 to 7680), and C (positions 7640 to 10808).

    Article Title: Features of autophagic cell death in Plasmodium liver-stage parasites
    Article Snippet: Genes coding for PbATG8 and ScATG8 were amplified from cDNA using primer pairs 5′- G GCGGCCGC ATGCCA TCATTAAAAG-3′ and 5′- GG GCGGCCGC TTATCCAAAACAACTTTC3′ and 5′-G GCGGCCGC CTACCTGCCAAATGTATTTTC-3′ and 5′-GG GCGGCCGC TAGCCTACCTGC CAAATGTAT TTTC-3, respectively. .. The fragments were cloned into the yeast expression vector pFL61 via NotI (New England Biolabs, R0189) and the resulting plasmids used to transform Scatg8Δ WCG strains of S. cerevisiae by the acetate method. .. In addition, the Scatg8Δ WCG strain and the wild-type WCG strain were transformed with the empty vector.

    Labeling:

    Article Title: ATP signaling in the integrative neural center of Aplysia californica
    Article Snippet: The two isoforms of AcP2X vary by a 147 base deletion/insertion and would be difficult to distinguishable by in situ. .. The antisense probe was generated by digestion of the AcP2X plasmid with Not I (CatR0189s, New England Biolabs Inc.) then transcribed with T3 polymerase from the DIG RNA Labeling Kit (Cat#11175025910, Roche Diagnostics). .. The control sense probe was produced by the same protocol but used Pme1 (Cat# R0560s, New England Biolabs Inc.) for digestion and T7 polymerase for transcription.

    Article Title: ATP signaling in the integrative neural center of Aplysia californica
    Article Snippet: The two isoforms of Ac P2X vary by a 147 base deletion/insertion and would not be distinguishable by in situ . .. The antisense probe was generated by digestion of the AcP2X plasmid with Not I (CatR0189s, New England Biolabs Inc.) then transcribed with T3 polymerase from the DIG RNA Labeling Kit (Cat#11175025910, Roche Diagnostics). .. The control sense probe was produced by the same protocol but used Pme1 (Cat# R0560s, New England Biolabs Inc.) for digestion and T7 polymerase for transcription.

    Low Copy Number:

    Article Title: N-glycosylation of Viral E Protein Is the Determinant for Vector Midgut Invasion by Flaviviruses
    Article Snippet: .. All of the segments were further assembled into low-copy-number vector PACYC177 with a T7 promoter in front of the 5′ terminus and a NotI restriction site following the 3′ end using NEBuilder Hi-Fi DNA assembly master mix (NEB Biolabs). .. The Natal-RGN strain was synthesized using sequence as the template and divided into three pieces, including A (positions 1 to 3370), B (positions 3325 to 7680), and C (positions 7640 to 10808).

    Clone Assay:

    Article Title: Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation
    Article Snippet: Finally, 32 copies of α 21-ITetO 11-mer has been cut out from the BAC vector and ligated into the same vector of α 21-IILacO/Gal4 12-mer to obtain the final product, pBAC11.32TW12.32GLII. .. After each cloning step, the forming arrays were digested with BamHI and NotI restriction enzymes (NEB) and analysed on 1% agarose gel electrophoresis using 100 bp DNA Ladder, Quick-Load 1 kb Extend DNA ladder or Low Range PFG Marker (NEB). .. Quantitative PCR (qPCR) to detect BAC copy numberCells from HAC-containing clones have been harvested and genomic DNA has been collected using Maxwell DNA purification kit (Promega). qPCR analysis have been performed using SYBR Green Master Mix (Roche) and the following primers: N11F5: 5’-GGGATCACTAGCAATAAAAGGTAGAC-3’ and N11R6: 5’-TCCTTCTGTCTCGTTTTTATGGC-3’ for the BAC synthetic DNA; 11-10R: 5’-AGG GAA TGT CTT CCC ATA AAA ACT-3’and mCbox-4: 5’-GTC TAC CTT TTA TTT GAA TTC CCG-3’ for the alphoid chr21 array as control.

    Article Title: Features of autophagic cell death in Plasmodium liver-stage parasites
    Article Snippet: Genes coding for PbATG8 and ScATG8 were amplified from cDNA using primer pairs 5′- G GCGGCCGC ATGCCA TCATTAAAAG-3′ and 5′- GG GCGGCCGC TTATCCAAAACAACTTTC3′ and 5′-G GCGGCCGC CTACCTGCCAAATGTATTTTC-3′ and 5′-GG GCGGCCGC TAGCCTACCTGC CAAATGTAT TTTC-3, respectively. .. The fragments were cloned into the yeast expression vector pFL61 via NotI (New England Biolabs, R0189) and the resulting plasmids used to transform Scatg8Δ WCG strains of S. cerevisiae by the acetate method. .. In addition, the Scatg8Δ WCG strain and the wild-type WCG strain were transformed with the empty vector.

    Agarose Gel Electrophoresis:

    Article Title: Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation
    Article Snippet: Finally, 32 copies of α 21-ITetO 11-mer has been cut out from the BAC vector and ligated into the same vector of α 21-IILacO/Gal4 12-mer to obtain the final product, pBAC11.32TW12.32GLII. .. After each cloning step, the forming arrays were digested with BamHI and NotI restriction enzymes (NEB) and analysed on 1% agarose gel electrophoresis using 100 bp DNA Ladder, Quick-Load 1 kb Extend DNA ladder or Low Range PFG Marker (NEB). .. Quantitative PCR (qPCR) to detect BAC copy numberCells from HAC-containing clones have been harvested and genomic DNA has been collected using Maxwell DNA purification kit (Promega). qPCR analysis have been performed using SYBR Green Master Mix (Roche) and the following primers: N11F5: 5’-GGGATCACTAGCAATAAAAGGTAGAC-3’ and N11R6: 5’-TCCTTCTGTCTCGTTTTTATGGC-3’ for the BAC synthetic DNA; 11-10R: 5’-AGG GAA TGT CTT CCC ATA AAA ACT-3’and mCbox-4: 5’-GTC TAC CTT TTA TTT GAA TTC CCG-3’ for the alphoid chr21 array as control.

    Marker:

    Article Title: Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation
    Article Snippet: Finally, 32 copies of α 21-ITetO 11-mer has been cut out from the BAC vector and ligated into the same vector of α 21-IILacO/Gal4 12-mer to obtain the final product, pBAC11.32TW12.32GLII. .. After each cloning step, the forming arrays were digested with BamHI and NotI restriction enzymes (NEB) and analysed on 1% agarose gel electrophoresis using 100 bp DNA Ladder, Quick-Load 1 kb Extend DNA ladder or Low Range PFG Marker (NEB). .. Quantitative PCR (qPCR) to detect BAC copy numberCells from HAC-containing clones have been harvested and genomic DNA has been collected using Maxwell DNA purification kit (Promega). qPCR analysis have been performed using SYBR Green Master Mix (Roche) and the following primers: N11F5: 5’-GGGATCACTAGCAATAAAAGGTAGAC-3’ and N11R6: 5’-TCCTTCTGTCTCGTTTTTATGGC-3’ for the BAC synthetic DNA; 11-10R: 5’-AGG GAA TGT CTT CCC ATA AAA ACT-3’and mCbox-4: 5’-GTC TAC CTT TTA TTT GAA TTC CCG-3’ for the alphoid chr21 array as control.

    Expressing:

    Article Title: Features of autophagic cell death in Plasmodium liver-stage parasites
    Article Snippet: Genes coding for PbATG8 and ScATG8 were amplified from cDNA using primer pairs 5′- G GCGGCCGC ATGCCA TCATTAAAAG-3′ and 5′- GG GCGGCCGC TTATCCAAAACAACTTTC3′ and 5′-G GCGGCCGC CTACCTGCCAAATGTATTTTC-3′ and 5′-GG GCGGCCGC TAGCCTACCTGC CAAATGTAT TTTC-3, respectively. .. The fragments were cloned into the yeast expression vector pFL61 via NotI (New England Biolabs, R0189) and the resulting plasmids used to transform Scatg8Δ WCG strains of S. cerevisiae by the acetate method. .. In addition, the Scatg8Δ WCG strain and the wild-type WCG strain were transformed with the empty vector.

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    • yeast expression vector pfl61  (New England Biolabs)
    98
    New England Biolabs yeast expression vector pfl61
    Figure 7. PbAtg8 does not localize to autophagosomes in dying parasites ( A ) and cannot complement yeast Atg8 ( B ). ( A ) HepG2 cells were infected with P. berghei parasites constitutively expressing mCherry (mCherry). 60 hpi the cells were fixed and stained with an anti-PbACP antiserum (ACP) to label the apicoplast and an anti-PbAtg8 antiserum (PbAtg8) to monitor the localization of this protein during parasite cell death. DNA was labeled with DAPI. A representative parasite showing strong vacuolization is depicted. A higher magnification of some important details is presented in the merged image. Scale bar: 10 µm: CPS. ( B ) Scatg8Δ and wt strains were transformed with empty <t>pFL61</t> plasmid or the same plasmid containing PbAtg8 or ScAtg8 as a positive control. Western blot analysis was performed with the transformed strains using anti-aminopeptidase I antibodies. Transport of prApe1 to the vacuole where it matures (Ape1) only takes place in the presence of a functional autophagy pathway. The prApe1 and the Ape1 bands are marked with arrows.
    Yeast Expression Vector Pfl61, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/yeast expression vector pfl61/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
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    Figure 7. PbAtg8 does not localize to autophagosomes in dying parasites ( A ) and cannot complement yeast Atg8 ( B ). ( A ) HepG2 cells were infected with P. berghei parasites constitutively expressing mCherry (mCherry). 60 hpi the cells were fixed and stained with an anti-PbACP antiserum (ACP) to label the apicoplast and an anti-PbAtg8 antiserum (PbAtg8) to monitor the localization of this protein during parasite cell death. DNA was labeled with DAPI. A representative parasite showing strong vacuolization is depicted. A higher magnification of some important details is presented in the merged image. Scale bar: 10 µm: CPS. ( B ) Scatg8Δ and wt strains were transformed with empty pFL61 plasmid or the same plasmid containing PbAtg8 or ScAtg8 as a positive control. Western blot analysis was performed with the transformed strains using anti-aminopeptidase I antibodies. Transport of prApe1 to the vacuole where it matures (Ape1) only takes place in the presence of a functional autophagy pathway. The prApe1 and the Ape1 bands are marked with arrows.

    Journal: Autophagy

    Article Title: Features of autophagic cell death in Plasmodium liver-stage parasites

    doi: 10.4161/auto.23689

    Figure Lengend Snippet: Figure 7. PbAtg8 does not localize to autophagosomes in dying parasites ( A ) and cannot complement yeast Atg8 ( B ). ( A ) HepG2 cells were infected with P. berghei parasites constitutively expressing mCherry (mCherry). 60 hpi the cells were fixed and stained with an anti-PbACP antiserum (ACP) to label the apicoplast and an anti-PbAtg8 antiserum (PbAtg8) to monitor the localization of this protein during parasite cell death. DNA was labeled with DAPI. A representative parasite showing strong vacuolization is depicted. A higher magnification of some important details is presented in the merged image. Scale bar: 10 µm: CPS. ( B ) Scatg8Δ and wt strains were transformed with empty pFL61 plasmid or the same plasmid containing PbAtg8 or ScAtg8 as a positive control. Western blot analysis was performed with the transformed strains using anti-aminopeptidase I antibodies. Transport of prApe1 to the vacuole where it matures (Ape1) only takes place in the presence of a functional autophagy pathway. The prApe1 and the Ape1 bands are marked with arrows.

    Article Snippet: The fragments were cloned into the yeast expression vector pFL61 via NotI (New England Biolabs, R0189) and the resulting plasmids used to transform Scatg8Δ WCG strains of S. cerevisiae by the acetate method.

    Techniques: Infection, Expressing, Staining, Labeling, Transformation Assay, Plasmid Preparation, Positive Control, Western Blot, Functional Assay

    Distribution of AcP2X in the CNS of Aplysia and the effect of ATP on Aplysia F-cluster neurons. ( A , B ) AcP2X is expressed in neurons of the cerebral F-cluster (in situ hybridization). A pair of giant serotonergic feeding interneurons (MCC) are indicated by arrows. ( A ) The preparation embedded in a mounting media. ( B ) The cerebral ganglion was photographed in 100% ethanol. Scale 300 µm. ( C ) Current-clamp recording from F-cluster neurons in the intact CNS. Bath application of ATP (2.0 mM) caused an excitatory response with spiking activity (2–5 mV depolarization with a burst of the action potentials), and full recovery following washout (indicated by arrows) (n = 7, neurons). ( D ) Voltage-clamp recording from F-cluster neuron. Raw traces were recorded in response to 2.0 mM of ATP at three holding potentials (agonist application indicated by the line).

    Journal: Scientific Reports

    Article Title: ATP signaling in the integrative neural center of Aplysia californica

    doi: 10.1038/s41598-021-84981-5

    Figure Lengend Snippet: Distribution of AcP2X in the CNS of Aplysia and the effect of ATP on Aplysia F-cluster neurons. ( A , B ) AcP2X is expressed in neurons of the cerebral F-cluster (in situ hybridization). A pair of giant serotonergic feeding interneurons (MCC) are indicated by arrows. ( A ) The preparation embedded in a mounting media. ( B ) The cerebral ganglion was photographed in 100% ethanol. Scale 300 µm. ( C ) Current-clamp recording from F-cluster neurons in the intact CNS. Bath application of ATP (2.0 mM) caused an excitatory response with spiking activity (2–5 mV depolarization with a burst of the action potentials), and full recovery following washout (indicated by arrows) (n = 7, neurons). ( D ) Voltage-clamp recording from F-cluster neuron. Raw traces were recorded in response to 2.0 mM of ATP at three holding potentials (agonist application indicated by the line).

    Article Snippet: The antisense probe was generated by digestion of the AcP2X plasmid with Not I (Cat#R0189s, New England Biolabs Inc.) then transcribed with T3 polymerase from the DIG RNA Labeling Kit (Cat#11175025910, Roche Diagnostics).

    Techniques: In Situ Hybridization, Activity Assay

    Generation of synthetic α 21-I TetO and α 21-II LacO/Gal4 arrays. (A) Scheme of the pBAC11.32TW12.32GLII containing BAC and YAC cassettes, G418 resistance cassette and synthetic DNA: α 21-I TetO formed by high ordered repeats (HOR) monomers (green arrows) containing CENP-B boxes (blue) alternate with monomers containing TetO (yellow); α 21-II LacO/Gal4 formed by high ordered repeats (HOR) monomers (yellow arrows) containing Gal4 binding sequence (green) alternating with LacO (red). (B) Schematic of the assembly of the α 21-I TetO and α 21-II LacO/Gal4 arrays. (C, D) PFGE analysis of the nascent α 21-I TetO and α 21-II LacO/Gal4 arrays, cut with BamHI/NotI after each cycles of tandem ligation array amplification as described in Figure S2A (C) and Figure S2B (D). Expected sizes: α 21-I TetO 11-mer 1 copy (1.9 kb), 8 copies (15.2 kb), 32 copies (60.8 kb); α 21-II LacO/Gal4 12-mer 1 copy (2 kb), 8 copies (16 kb), 32 copies (64 kb). Plasmid vector is 2.9 kb, BAC vector is 7.1 kb. The asterisk (*) indicates the fragments that have been cloned into BAC vector (8 copies, 16 kb); red arrow in D indicates the size of the final pBAC11.32TW12.32GLII (∼120 kb) (m and M, markers).

    Journal: bioRxiv

    Article Title: Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

    doi: 10.1101/2020.07.02.184408

    Figure Lengend Snippet: Generation of synthetic α 21-I TetO and α 21-II LacO/Gal4 arrays. (A) Scheme of the pBAC11.32TW12.32GLII containing BAC and YAC cassettes, G418 resistance cassette and synthetic DNA: α 21-I TetO formed by high ordered repeats (HOR) monomers (green arrows) containing CENP-B boxes (blue) alternate with monomers containing TetO (yellow); α 21-II LacO/Gal4 formed by high ordered repeats (HOR) monomers (yellow arrows) containing Gal4 binding sequence (green) alternating with LacO (red). (B) Schematic of the assembly of the α 21-I TetO and α 21-II LacO/Gal4 arrays. (C, D) PFGE analysis of the nascent α 21-I TetO and α 21-II LacO/Gal4 arrays, cut with BamHI/NotI after each cycles of tandem ligation array amplification as described in Figure S2A (C) and Figure S2B (D). Expected sizes: α 21-I TetO 11-mer 1 copy (1.9 kb), 8 copies (15.2 kb), 32 copies (60.8 kb); α 21-II LacO/Gal4 12-mer 1 copy (2 kb), 8 copies (16 kb), 32 copies (64 kb). Plasmid vector is 2.9 kb, BAC vector is 7.1 kb. The asterisk (*) indicates the fragments that have been cloned into BAC vector (8 copies, 16 kb); red arrow in D indicates the size of the final pBAC11.32TW12.32GLII (∼120 kb) (m and M, markers).

    Article Snippet: After each cloning step, the forming arrays were digested with BamHI and NotI restriction enzymes (NEB) and analysed on 1% agarose gel electrophoresis using 100 bp DNA Ladder, Quick-Load 1 kb Extend DNA ladder or Low Range PFG Marker (NEB).

    Techniques: BAC Assay, Binding Assay, Sequencing, Ligation, Amplification, Plasmid Preparation, Clone Assay

    Screening of HT1080 colonies after transfection with pBAC11.32TW12.32GLII. (A) Scheme showing the possible fates of the pBAC11.32TW12.32GLII HAC seeding DNA after transfection in HT1080: in yellow and green (as integration or HAC) is represented the synthetic DNA. Timeline of the experiments performed from transfection into HT1080 cells. (B) BAC copy number (y axis) analyzed by qPCR in each HT1080 clone (x axis): only HT1080 clones containing > 20 BAC copies are represented in the graph. HT1080 clones are represented in green (HAC), red (integration) or mixture (both) according to the results of the FISH screening, as shown in C. Black arrows indicate the clones shown in C and analyzed further. (C) Representative pictures of oligo-FISH staining of HT1080 clones: slides have been hybridized with DNA probes (TetO-dig/rhodamine -dig antibody, Gal4-biotin and LacO-biotin/Fitc-streptavidin). DAPI stains DNA. Scalebar = 10µm. (D) Southern blot of selected HT1080 clonal DNA (as labelled on top of the panel) digested with BamHI and separated by CHEF; the transferred membrane was hybridized with radioactively labelled TetO (left) or LacO (right) specific probes. Red arrows indicate the expected size of the band without rearrangements. Clones labelled in red have been screened further (M and m, markers). (E) Cartoon of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI.

    Journal: bioRxiv

    Article Title: Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

    doi: 10.1101/2020.07.02.184408

    Figure Lengend Snippet: Screening of HT1080 colonies after transfection with pBAC11.32TW12.32GLII. (A) Scheme showing the possible fates of the pBAC11.32TW12.32GLII HAC seeding DNA after transfection in HT1080: in yellow and green (as integration or HAC) is represented the synthetic DNA. Timeline of the experiments performed from transfection into HT1080 cells. (B) BAC copy number (y axis) analyzed by qPCR in each HT1080 clone (x axis): only HT1080 clones containing > 20 BAC copies are represented in the graph. HT1080 clones are represented in green (HAC), red (integration) or mixture (both) according to the results of the FISH screening, as shown in C. Black arrows indicate the clones shown in C and analyzed further. (C) Representative pictures of oligo-FISH staining of HT1080 clones: slides have been hybridized with DNA probes (TetO-dig/rhodamine -dig antibody, Gal4-biotin and LacO-biotin/Fitc-streptavidin). DAPI stains DNA. Scalebar = 10µm. (D) Southern blot of selected HT1080 clonal DNA (as labelled on top of the panel) digested with BamHI and separated by CHEF; the transferred membrane was hybridized with radioactively labelled TetO (left) or LacO (right) specific probes. Red arrows indicate the expected size of the band without rearrangements. Clones labelled in red have been screened further (M and m, markers). (E) Cartoon of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI.

    Article Snippet: After each cloning step, the forming arrays were digested with BamHI and NotI restriction enzymes (NEB) and analysed on 1% agarose gel electrophoresis using 100 bp DNA Ladder, Quick-Load 1 kb Extend DNA ladder or Low Range PFG Marker (NEB).

    Techniques: Transfection, HAC Assay, BAC Assay, Real-time Polymerase Chain Reaction, Clone Assay, Fluorescence In Situ Hybridization, Staining, Southern Blot

    Formation of input pBAC11.32TW12.32GLII DNA. (A) CHEF analysis of 16 bacterial DNA after transformation with pBAC11.32TW12.32GLII and NotI and BamHI digestion: red arrows indicate the size of the final vector (∼120 kb); colonies labelled in red contain the insert of the desired length. DNA used for transfection as a control (in duplicate) (M marker). (B) Scheme of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI used to release the synthetic DNA. (C) PFGE analysis of selected bacterial colonies (in red) digested with EcoRI: each fragment originates from a different array (label on the left). DNA used for transfection as a control (in duplicate); original DNA as uncut sample (M marker). (D) α 21-I TetO and α 21-II LacO/Gal4 DNA ratio calculated with ImageJ on the intensity of the bands shown in C for each bacterial colony. Control and original DNA as in C. (E) CHEF analysis of bacterial colony #1 DNA (in duplicate) digested with NotI and BamHI to release the synthetic DNA (m and M, markers).

    Journal: bioRxiv

    Article Title: Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

    doi: 10.1101/2020.07.02.184408

    Figure Lengend Snippet: Formation of input pBAC11.32TW12.32GLII DNA. (A) CHEF analysis of 16 bacterial DNA after transformation with pBAC11.32TW12.32GLII and NotI and BamHI digestion: red arrows indicate the size of the final vector (∼120 kb); colonies labelled in red contain the insert of the desired length. DNA used for transfection as a control (in duplicate) (M marker). (B) Scheme of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI used to release the synthetic DNA. (C) PFGE analysis of selected bacterial colonies (in red) digested with EcoRI: each fragment originates from a different array (label on the left). DNA used for transfection as a control (in duplicate); original DNA as uncut sample (M marker). (D) α 21-I TetO and α 21-II LacO/Gal4 DNA ratio calculated with ImageJ on the intensity of the bands shown in C for each bacterial colony. Control and original DNA as in C. (E) CHEF analysis of bacterial colony #1 DNA (in duplicate) digested with NotI and BamHI to release the synthetic DNA (m and M, markers).

    Article Snippet: After each cloning step, the forming arrays were digested with BamHI and NotI restriction enzymes (NEB) and analysed on 1% agarose gel electrophoresis using 100 bp DNA Ladder, Quick-Load 1 kb Extend DNA ladder or Low Range PFG Marker (NEB).

    Techniques: Transformation Assay, Plasmid Preparation, Transfection, Marker

    Distribution of AcP2X in the CNS of Aplysia and the effect of ATP on Aplysia F-cluster neurons. A and B : AcP2X is expressed in neurons of the cerebral F-cluster ( in situ hybridization). A pair of giant serotonergic feeding interneurons (MCC) are indicated by arrows. A . The preparation embedded in a mounting media. B . The cerebral ganglion was photographed in 100% ethanol. Scale: 300 μm. C . Current-clamp recording from F-cluster neurons in the intact CNS. Bath application of ATP (2.0 mM) caused an excitatory response with spiking activity (2-5 mV depolarization with a burst of the action potentials), and full recovery following washout (indicated by arrows). D . Voltage-clamp recording from F-cluster neuron. Raw traces recorded in response to 2.0 mM of ATP at three holding potentials (agonist application indicated by the line).

    Journal: bioRxiv

    Article Title: ATP signaling in the integrative neural center of Aplysia californica

    doi: 10.1101/2020.11.20.392001

    Figure Lengend Snippet: Distribution of AcP2X in the CNS of Aplysia and the effect of ATP on Aplysia F-cluster neurons. A and B : AcP2X is expressed in neurons of the cerebral F-cluster ( in situ hybridization). A pair of giant serotonergic feeding interneurons (MCC) are indicated by arrows. A . The preparation embedded in a mounting media. B . The cerebral ganglion was photographed in 100% ethanol. Scale: 300 μm. C . Current-clamp recording from F-cluster neurons in the intact CNS. Bath application of ATP (2.0 mM) caused an excitatory response with spiking activity (2-5 mV depolarization with a burst of the action potentials), and full recovery following washout (indicated by arrows). D . Voltage-clamp recording from F-cluster neuron. Raw traces recorded in response to 2.0 mM of ATP at three holding potentials (agonist application indicated by the line).

    Article Snippet: The antisense probe was generated by digestion of the AcP2X plasmid with Not I (Cat#R0189s, New England Biolabs Inc.) then transcribed with T3 polymerase from the DIG RNA Labeling Kit (Cat#11175025910, Roche Diagnostics).

    Techniques: In Situ Hybridization, Activity Assay