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Home > Products > Polymerases > Mesophilic DNA Polymerases > DNA Polymerase I, Large (Klenow) Fragment > FAQ DNA Polymerase I, Large (Klenow) Fragment FAQ See the Polymerases FAQ also. 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 75°C 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 25°C *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 25°C or room temperature, but T4 DNA Polymerase must be used at 12°C 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.