M0210 FAQs for DNA Polymerase I, Large (Klenow) Fragment, Mesophilic DNA Polymerases, Intl, NEB
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DNA Polymerase I, Large (Klenow) Fragment FAQ

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

Q1: Can DNA Polymerase I, Large (Klenow) Fragment be used in other NEBuffers?

A1: It is active in all four NEBuffers and T4 DNA Ligase Buffer, but optimal activity is observed in NEBuffer 2. All NEBuffers must be supplemented with dNTPs.


Q2: Can DNA Polymerase I, Large (Klenow) Fragment be used to blunt DNA?

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


Q3: Can DNA Polymerase I, Large (Klenow) Fragment 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 DNA Polymerase I, Large (Klenow) Fragment be used to chew back 5' overhangs?

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


Q5: Are NEB DNA Polymerases supplied with dNTPs?

A5: 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.


Q6: Can DNA Polymerase I, Large (Klenow) Fragment be heat inactivated?

A6: 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.


Q7: Are the nucleotides needed to remove a 3' overhang with DNA Polymerase I, Large (Klenow) Fragment?

A7: Yes, in the absence of dNTPs the enzyme will remove more nucleotides than necessary to blunt.


Q8: What are the main causes for blunting reaction failure using DNA Polymerase I, Large (Klenow) Fragment?

A8: 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 temperatures greater than 25C
*Failure to add nucleotides or the nucleotide level is too low
*Heat inactivating without EDTA



Q9: Can DNA Polymerase I, Large (Klenow) Fragment be used in labeling reactions and partial fill in reactions?

A9: The 3'→5' exonuclease of DNA Polymerase I, Large (Klenow) Fragment makes these reactions difficult to control so Klenow Fragment (3'→5') exo- (
NEB# M0212) is recommended instead.


Q10: Is DNA Polymerase I, Large (Klenow) Fragment the enzyme of choice for chewing back 3' overhangs and filling in 5' overhangs (3' recessed ends)?

A10: DNA Polymerase I, Large (Klenow) Fragment and T4 DNA Polymerase (
NEB# M0203) 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.