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Found 5 matching solutions for this experiment
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The reaction mixture was purified with the Qiagen PCR Purification Kit and incubated with 5 units of Klenow Fragment (3′ exo−) (NEB) for 30 minutes at 37 °C in 1x NEBuffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9) with 240 μM dATP (Promega) in a 50 μL final volume. 10 mM Tris-HCl, pH 8.5 was added to a volume of 90 μL and the reaction was incubated for 20 minutes at 75 °C to inactivate the enzyme before cooling to 12 °C. 300 fmol of “adapter1/2”, barcoded according to enzyme concentration, or 6 pmol of “adapter1/2” for the PvuI digest, were added to the reaction mixture, along with 10 ul 10x NEB T4 DNA Ligase Reaction Buffer (500 mM Tris-HCl, 100 mM MgCl2, 100 mM dithiothreitol, 10 mM ATP). |
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| Protocol tips |
| The reaction mixture was purified with the Qiagen PCR Purification Kit and incubated with 5 units of Klenow Fragment (3′ exo−) (NEB) for 30 minutes at 37 °C in 1x NEBuffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9) with 240 μM dATP (Promega) in a 50 μL final volume. 10 mM Tris-HCl, pH 8.5 was added to a volume of 90 μL and the reaction was incubated for 20 minutes at 75 °C to inactivate the enzyme before cooling to 12 °C. 300 fmol of “adapter1/2”, barcoded according to enzyme concentration, or 6 pmol of “adapter1/2” for the PvuI digest, were added to the reaction mixture, along with 10 ul 10x NEB T4 DNA Ligase Reaction Buffer (500 mM Tris-HCl, 100 mM MgCl2, 100 mM dithiothreitol, 10 mM ATP). |
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DNA replication in the aliquot containing [32P]∆N-His-PKA-LexA and [α32P]dATP was quenched for 10 min at 37 °C by adding an equal volume of stop buffer containing 50 mM HEPES-KOH (pH 8.0), 75 mM potassium glutamate, 10 mM Mg(OAc)2, 10 mM DTT, 100 μg/ml BSA, 4 mM AMP-PNP, 133 µM
2′,3′-dideoxyribonucleoside 5′-triphosphates, and
0.4 U/µl of each EcoRI-HF (NEB) and PvuI-HF
(NEB). EDTA was then added to a final concentration of 30 mM and the DNA products
analyzed by gel electrophoresis as described (17)
using 0.6% alkaline agarose and 0.8% native agarose gels followed by autoradiography and
phosphorimaging.
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| Protocol tips |
DNA replication in the aliquot containing [32P]∆N-His-PKA-LexA and [α32P]dATP was quenched for 10 min at 37 °C by adding an equal volume of stop buffer containing 50 mM HEPES-KOH (pH 8.0), 75 mM potassium glutamate, 10 mM Mg(OAc)2, 10 mM DTT, 100 μg/ml BSA, 4 mM AMP-PNP, 133 µM
2′,3′-dideoxyribonucleoside 5′-triphosphates, and
0.4 U/µl of each EcoRI-HF (NEB) and PvuI-HF
(NEB). EDTA was then added to a final concentration of 30 mM and the DNA products
analyzed by gel electrophoresis as described (17)
using 0.6% alkaline agarose and 0.8% native agarose gels followed by autoradiography and
phosphorimaging.
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Digestion of chromatin and DNA was performed. First, the chromatin was digested with the following FastDigest enzymes: AatII and PvuI (Thermo Scientific, Waltham, MA, USA). The restriction enzymes were defined by in silico analysis. Sites of digestion for AatII and PvuI are located in the nucleosomes, where DNA wraps around nucleosomes. After digestion with restriction enzymes (15 min, 37 °C) and enzymes’ inactivation (AatII, PvuI –80 °C, 5 min), the samples were incubated with Proteinase K (Qiagen, Hilden, Germany) and RNase A (Qiagen, Hilden, Germany) at 37 °C for 1 h. |
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| Protocol tips |
| Digestion of chromatin and DNA was performed. First, the chromatin was digested with the following FastDigest enzymes: AatII and PvuI (Thermo Scientific, Waltham, MA, USA). The restriction enzymes were defined by in silico analysis. Sites of digestion for AatII and PvuI are located in the nucleosomes, where DNA wraps around nucleosomes. After digestion with restriction enzymes (15 min, 37 °C) and enzymes’ inactivation (AatII, PvuI –80 °C, 5 min), the samples were incubated with Proteinase K (Qiagen, Hilden, Germany) and RNase A (Qiagen, Hilden, Germany) at 37 °C for 1 h. |
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An expression plasmid coding for the full-length XPG/XPF protein or one of the splice variants, e.g. pcDNA3.1 (+)XPF, was linearized using the PvuI restriction enzyme (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions. |
Successful linearization was analyzed on an agarose gel and the linearized plasmids were purified by ethanol precipitation. |
| Protocol tips |
| An expression plasmid coding for the full-length XPG/XPF protein or one of the splice variants, e.g. pcDNA3.1 (+)XPF, was linearized using the PvuI restriction enzyme (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions. |
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| Successful linearization was analyzed on an agarose gel and the linearized plasmids were purified by ethanol precipitation. |
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Finally, the resulting pEX12–tTA2S was digested with NotI and PvuI Takara) to obtain a 7,627 bp fragment (PmThy1.2–tTA2S–p(A)) for pronuclear injection NotI and PvuI (Takara) to obtain a 7,627 bp fragment (PmThy1.2–tTA2S–p(A)) for pronuclear injection (Fig. 1a). |
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| Protocol tips |
| Finally, the resulting pEX12–tTA2S was digested with NotI and PvuI Takara) to obtain a 7,627 bp fragment (PmThy1.2–tTA2S–p(A)) for pronuclear injection NotI and PvuI (Takara) to obtain a 7,627 bp fragment (PmThy1.2–tTA2S–p(A)) for pronuclear injection (Fig. 1a). |
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