New England Biolabs, M0203S, T4 DNA Polymerase

T4 DNA Polymerase catalyzes the synthesis of DNA in the 5´→ 3´ direction and requires the presence of template and primer. Related Categories DNA Manipulation Applications Blunting,, Polymerases for DNA Manipulation,, PCR Specification Unit Definition One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 37°C (5). Reaction Conditions 1X NEBuffer™ r2.1 1X NEBuffer™ r2.1 50 mM NaCl 10 mM Tris-HCl 10 mM MgCl2 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Activity in NEBuffers Storage Buffer 100 mM KPO4 1 mM DTT 50% Glycerol pH 6.5 @ 25°C Heat Inactivation 75°C for 20 minutes Molecular Weight Theoretical: 104000 daltons 5' - 3' Exonuclease No 3' - 5' Exonuclease Yes Strand Displacement No Unit Assay Conditions 1X NEBuffer 2.1, 33 µM dNTPs including [ 3 H]-dTTP, 70 µg/ml denatured herring sperm DNA. Error Rate ~ 1x10 -6 bases FAQ Q: Which DNA polymerase can I use to blunt DNA? A: Note: dNTPs must be added to blunting reactions We primarily recommend DNA Polymerase I, Large (Klenow) Fragment (M0210) for blunting. Optimal blunting activity is at room temperature (25°C). T4 DNA Polymerase (M0203) can also be used to blunt but should be used at 12°C to prevent against the formation of recessed ends due to its very strong 3'-5' exonuclease activity. For blunting at thermophilic temperatures, we recommend Vent (M0254) or Deep Vent (M0258) DNA Polymerase. While Q5 High-Fidelity Polymerase may also be used for blunting at thermophilic temperatures, it has very strict buffering requirements for optimal activity and should only be used with Q5 Reaction Buffer. Mung Bean Nuclease (M0250) is our only catalog enzyme that can remove 5' overhangs. For inactivation, we recommend heating + EDTA; please click here for our inactivation guidelines. Learn More DNA polymerases are often used to blunt by filling in protruding 5' ends (5´ → 3´ polymerization activity) and/or chewing back (3´ → 5´ exonuclease activity) 3' overhangs. In the absence of dNTPs, 3'-5' exonuclease activity will overwhelm the 5'-3' polymerase activity to remove more nucleotides than desired. However, 5'-3' polymerization activity is preferred by the enzyme compared to 3'-5' exonuclease activity in the presence of dNTPs. The presence of dNTPs ensures that nucleotides are filled back in across from the template, resulting in blunt ends. For more information, please see our other FAQs: What does exonuclease activity mean for a DNA polymerase? What ends will my PCR products have? Q: Can I use a DNA polymerase to fill in 3' overhangs or remove 5' overhangs? A: No, DNA polymerases cannot fill in 3' overhangs because they can only add nucleotides in the 5'-3' direction. To remove 3' overhangs, see our blunting FAQ. No, DNA polymerases cannot remove 5' overhangs. Mung Bean Nuclease (M0250) is the only catalog enzyme that can remove 5' overhangs. Q: How can I heat inactivate this DNA polymerase? A: For DNA Polymerase I (M0209), Large Klenow Fragment (M0210), T4 DNA Polymerase (M0203), and T7 DNA Polymerase (M0274): To prevent formation of recessed ends (past the desired blunting point), pre-heat a water bath or thermocycler to 75°C. Before placing the sample at 75°C, add EDTA to your sample to a final concentration of 10 mM (to chelate the Mg2+ cofactor) then incubate at 75°C for 20 minutes. Note that elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times will result in recessed ends due to the 3´ → 5´ exonuclease activity of the enzyme. Klenow Fragment (3'→5' exo-) Inactivate by heating at 75°C for 20 minutes. EDTA is not necessary due to the lack of 3'-5' exonuclease activity. Q: Can this DNA polymerase be used in labeling and partial fill reactions? A: For evenly labeled, full-length coverage, DNA Polymerase I (E. coli) (M0209) is the preferred enzyme. At elevated reaction temperatures, Taq DNA Polymerase may also be used. For high yield, short, fragmented (~250 bp) dsDNA generation, Klenow Fragment (3'→5' exo-) (M0212) is recommended. Nick Translation When using nick translation to generate probes, DNase I is typically used to nick the DNA. DNA Polymerase I (E. coli) removes leading bases at the nick with its 5'→3' exonuclease activity and then fills in with labeled bases. Modified Nucleotides Since incorporation efficiency of modified nucleotides is generally lower than their natural counterparts, a 100% replacement can cause the polymerase to stall. To balance yield with labeling efficiency, we generally recommend testing several ratios of modified to natural dNTP. The following polymerases are not recommended for labeling or partial fill reactions because their 3'-5' exonuclease activity is difficult to control: DNA Polymerase I, Large (Klenow) Fragment (M2010) T4 DNA Polymerase (M0203) T7 DNA Polymerase (M0274) Q: What are the main causes of blunting reaction failure? A: The following conditions can cause exonuclease activity to overwhelm the polymerase activity, causing recessed instead of blunt ends: Failure to add nucleotides, or the nucleotide concentration is too low Adding too much enzyme Incubating for too long Incubating at temperatures greater than the recommended temperature Heat inactivating without EDTA FAQ: Can this polymerase be heat inactivated? Please see the recommended blunting protocol for DNA Polymerase I, Large (Klenow) Fragment by clicking here. T4 DNA Polymerase by clicking here. Q: Can this DNA polymerase be used in other NEBuffers for filling in or blunting overhangs? A: FOR FILLING IN 5' OVERHANGS DNA Polymerase I (E. coli), Large Klenow Fragment, Klenow Fragment (3'-5' exo-), and T4 DNA polymerase are all active in all NEBuffers, but NEBuffer 2/2.1/r2.1 provides optimal activity. Optimal activity of T7 DNA Polymerase is seen in its own buffer, and it has 50% reduced activity in the other NEBuffers. For comparisons of NEBuffer compositions, click here. To see the activity of more DNA modifying enzymes in rCutSmart Buffer, click here. FOR BLUNTING OVERHANGS NEBuffers DNA Polymerase I (E. coli) DNA Polymerase I, Large (Klenow) Fragment T4 DNA Polymerase T7 DNA Polymerase 1 Y N Y 50% 1.1 Y N Y 50% r1.1 Y N Y 50% 2 Optimal Optimal Y 50% 2.1 Y Y Y 50% r2.1 Y Y Optimal 50% 3 Y Y N 50% 3.1 Y Y N 50% r3.1 Y Y N 50% 4 Y Y Y 50% CutSmart Y Y Y 50% rCutSmart Y Y Y 50% T4 DNA Ligase Buffer Y Y T7 DNA Polymerse Reaction Buffer Optimal "Y" means yes, this polymerase can be used in the specified buffer. "N" means no, this polymerase cannot be used in the specified buffer. Optimal buffers are indicated. Klenow (3'-5' exo-) cannot be used to remove overhangs.