New England Biolabs, M0269L, phi29 DNA Polymerase

Perfect for multiple displacement amplification Related Categories Isothermal Amplification & Strand Displacement Applications Whole Genome Amplification & Multiple Displacement Amplification,, Isothermal Amplification Specification Unit Definition One unit is defined as the amount of enzyme that will incorporate 0.5 pmol of dNTP into acid insoluble material in 10 minutes at 30°C. Reaction Conditions 1X phi29 DNA Polymerase Reaction Buffer Supplement with Recombinant Albumin, Molecular Biology Grade Incubate at 30°C 1X phi29 DNA Polymerase Reaction Buffer 50 mM Tris-HCl 10 mM MgCl2 10 mM (NH4)2SO4 4 mM DTT (pH 7.5 @ 25°C) Storage Buffer 10 mM Tris-HCl 100 mM KCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 0.5% Tween® 20 0.5% IGEPAL® CA-630 pH 7.4 @ 25°C Heat Inactivation 65°C for 10 minutes Molecular Weight Theoretical: 67000 daltons 5' - 3' Exonuclease No 3' - 5' Exonuclease Yes Strand Displacement ++++ Unit Assay Conditions 1X phi29 DNA Polymerase Reaction Buffer, 0.1 mg/ml Recombinant Albumin, 0.01 mg/ml HindIII-digested λ DNA, 0.2 µM dTTP including [ 3 H]-dTTP, 0.2 mM dGTP, 0.2 mM dATP and 0.2 mM dCTP. FAQ Q: Can phi29 DNA Polymerase be used in other NEBuffers? A: Yes. Phi29 DNA Polymerase is 30% active in NEBuffer 1, 35% active in NEBuffer 2, 25% active in NEBuffer 3, 90% active in NEBuffer 4, and 30% active in ThermoPol Buffer. It also is 100% active in rCutSmart buffer with the addition of 4mM DTT (final concentration). Q: Can phi29 DNA Polymerase be used to blunt DNA? A: It produces blunt-end fragments during DNA synthesis, so in theory it could be used to blunt DNA. Traditionally, DNA Polymerase I, Large (Klenow) Fragment (NEB# M0210) or T4 DNA Polymerase (NEB# M0203) are used to blunt DNA. Q: Can phi29 DNA Polymerase be used to fill in 3' overhangs? A: No, DNA polymerases cannot fill in 3' overhangs. To create blunt ends 3' overhangs must be removed. DNA Polymerase I, Large (Klenow) Fragment (NEB# M0210) or T4 DNA Polymerase (NEB# M0203) are the best choices to remove 3' overhangs. Mung Bean Nuclease (NEB# M0250) can also be used to remove 3´ overhangs. Q: Can phi29 DNA Polymerase be used to remove 5' overhangs? A: No, DNA polymerases cannot remove 5' overhangs. Use Mung Bean Nuclease (NEB# M0250) to remove 5' overhangs. Q: Can phi29 DNA Polymerase be heat inactivated? A: Yes, heat at 65°C for 10 minutes. Q: At what rate does phi29 DNA Polymerase add nucleotides to a primed single-stranded template? A: The following are phi29 DNA Polymerase rates at different temperatures using a primed single-stranded M13 template(1): 2280 nt/min at 30°C 1490 nt/min at 15°C 290 nt/mn at 4°C (1) Soengas, M.S., Gutierrez, M.S. and Salas, M. 1995 J. Mol. Biol.253(4):517-529. Q: Will phi29 DNA Polymerase work in rCutSmart buffer? A: Yes, phi29 DNA Polymerase will work in rCutSmart buffer. To see its % functional activity in rCutSmart, and that of other DNA Modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in rCutSmart® Buffer chart. Q: Are NEB DNA Polymerases supplied with dNTPs? A: No, the dNTPs must be ordered separately. They can be ordered as a convenient mix (Deoxynucleotide (dNTP) Solution Mix (NEB# N0447) with a 10 mM concentration of each deoxynucleotide) or as a set of 4 individual tubes (Deoxynucleotide (dNTP) Solution Set (NEB# N0446) with a 100mM concentration of each deoxynucleotide).