M0203 FAQs for T4 DNA Polymerase, Mesophilic DNA Polymerases, Intl, NEB
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T4 DNA Polymerase FAQ

Q1: Can T4 DNA Polymerase be used in other NEBuffers?
Q2: Can T4 DNA Polymerase be used to blunt DNA?
Q3: Can T4 DNA Polymerase be used to fill in 3' overhangs?
Q4: Can T4 DNA Polymerase be used to remove 5' overhangs?
Q5: Can T4 DNA Polymerase be heat inactivated?
Q6: Are the nucleotides needed to remove a 3' overhang using T4 DNA Polymerase?
Q7: What are the main causes of blunting reaction failure using T4 DNA Polymerase?
Q8: Are NEB DNA Polymerases supplied with dNTPs?
Q9: Can T4 DNA Polymerase be used in labeling reactions and partial fill in reactions?
Q10: Is T4 DNA Polymerase active at room temperature?
Q11: Is T4 DNA Polymerase the enzyme of choice for removing 3' overhangs and filling in 5' overhangs (3' recessed ends)?

Q1: Can T4 DNA Polymerase be used in other NEBuffers?

A1: Optimal activity is seen in NEBuffer 2. Greater than 75% activity is seen in all NEB restriction buffers, including uniques. Also active in T4 DNA Ligase buffer.


Q2: Can T4 DNA Polymerase be used to blunt DNA?

A2: Yes, 5' overhangs (3' recessed ends) can be filled in and 3' overhangs will be removed.


Q3: Can T4 DNA Polymerase be used to fill in 3' overhangs?

A3: 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), T4 DNA Polymerase (NEB# M0203) or Mung Bean Nuclease (NEB# M0250) are the best choices to remove 3' overhangs.


Q4: Can T4 DNA Polymerase be used to remove 5' overhangs?

A4: No, DNA polymerases cannot remove 5' overhangs. Use Mung Bean Nuclease (
NEB# M0250) to remove 5' overhangs.


Q5: Can T4 DNA Polymerase be heat inactivated?

A5: Yes. Add 10 mM EDTA to chelate the Mg2+ cofactor, which protects the DNA ends as they "breathe" while the temperature is increased. Then heat at 75C for 20 minutes.


Q6: Are the nucleotides needed to remove a 3' overhang using T4 DNA Polymerase?

A6: Yes, T4 DNA Polymerase will remove bases past blunt if the nucleotide concentration is less than 100 μM.


Q7: What are the main causes of blunting reaction failure using T4 DNA Polymerase?

A7: The following conditions can cause the exonuclease to overwhelm the polymerase activity causing recessed instead of blunt ends:
*Adding too much enzyme
*Incubating for too long
*Incubating at greater than 12C
*Failure to add nucleotides or the nucleotide level is too low
*Heat inactivating without EDTA


Q8: Are NEB DNA Polymerases supplied with dNTPs?

A8: No, the dNTPs must be ordered separately. They can be ordered as a convenient mix, Deoxynucleotide Solution Mix (
NEB# N0447) with a 10 mM concentration of each nucleotide or as a set of 4 individual tubes, Deoxynucleotide Solution Set (NEB# N0446) with a 100mM concentration of each nucleotide.


Q9: Can T4 DNA Polymerase be used in labeling reactions and partial fill in reactions?

A9: The strong 3'→5' exonuclease of T4 DNA Polymerase makes these reactions difficult to control. We recommend Klenow Fragment (3'→5' exo-) (
NEB# M0212) for these applications.


Q10: Is T4 DNA Polymerase active at room temperature?

A10: We suggest 12C. The DNA ends "breathe" at higher temperatures allowing the exonuclease to remove nucleotides past blunt.


Q11: Is T4 DNA Polymerase the enzyme of choice for removing 3' overhangs and filling in 5' overhangs (3' recessed ends)?

A11: DNA Polymerase I, Large (Klenow) Fragment (
NEB# M0210) and T4 DNA Polymerase are the best choices for this application. DNA Polymerase I, Large (Klenow) Fragment can be used at 25C or room temperature, but T4 DNA Polymerase must be used at 12C due to its robust exonuclease. Both work well in a wide variety of buffers. Vent DNA Polymerase (NEB# M0254) and Deep Vent DNA Polymerase (NEB# M0258) can also be used but ThermoPol buffer must be used, the reaction temperature is high, and the enzyme cannot be heat inactivated. Mung Bean Nuclease (NEB# M0250) will chew back 3' overhangs but the strong exonuclease activity combined with the lack of polymerase activity yield a lower percentage of blunt ends.