PvuI-HF®

Protocol tips

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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.

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.

Publication protocol

" Reaction mixtures (10 µl) containing DNA
template (2 nM), 50 mM HEPES-KOH (pH 8.0), 75
mM potassium glutamate, 10 mM Mg(OAc)2, 10
mM DTT, 100 μg/ml BSA (NEB), 200 μM CTP,
UTP, and GTP, 40 μM dNTPs, 1 mM ATP, 100 nM
Tus, 1 μM SSB (monomer), 140 nM DnaA, 200 nM
DnaB (monomer), 200 nM DnaC, 320 nM DnaG,
12.5 nM HU, 30 nM β, 20 nM Pol III*, 20 nM
gyrase, 2 µM RecA, 25 nM RecF, 200 nM RecO,
500 nM RecR, and 3 µM [32P]∆N-His-PKA-LexA were divided in half. [α32P]dATP was added to one
half and an equivalent volume of H2O to the other
half. Reaction mixtures were incubated at 37 °C for
either 8 or 20 min, as specified. The aliquot
containing only [
32P]∆N-His-PKA-LexA was
mixed with 2x SDS loading dye and analyzed as
described in the previous section. 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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