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Klenow Fragment (3´→5´ exo–) > FAQ |  | Klenow Fragment (3´→5´ exo–) FAQ
See the Polymerases FAQ also.
Q1: Can Klenow Fragment (3'→5' exo-) be used in other NEBuffers?
Q2: Can Klenow Fragment (3'→5' exo-) be used to blunt DNA?
Q3: Can Klenow Fragment (3'→5' exo-) be used to fill in 3' overhangs?
Q4: Can Klenow Fragment (3'→5' exo-) be used to remove 5' overhangs?
Q5: Can Klenow Fragment (3'→5' exo-) be heat inactivated?
Q6: Can Klenow Fragment (3'→5' exo-) be used in labeling reactions and partial fill in reactions?
Q7: Are NEB DNA Polymerases supplied with dNTPs?
Q8: Are there other methods for making probes?
Q9: What is the enzyme of choice for chewing back 3' overhangs and filling in 5' overhangs (3' recessed ends)?
Q1: Can Klenow Fragment (3'→5' exo-) be used in other NEBuffers?
A1: It is active in all four NEBuffers, but optimal activity is observed in NEBuffer 2. All NEBuffers must be supplemented with dNTPs.
Q2: Can Klenow Fragment (3'→5' exo-) be used to blunt DNA?
A2: No, 5' overhangs (3' recessed ends) can be filled in but the enzyme tends to leave a single base overhang (30-50%).
Q3: Can Klenow Fragment (3'→5' exo-) 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 Klenow Fragment (3'→5' exo-) 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 Klenow Fragment (3'→5' exo-) be heat inactivated?
A5: Yes, heat at 75°C for 20 minutes.
Q6: Can Klenow Fragment (3'→5' exo-) be used in labeling reactions and partial fill in reactions?
A6: Yes, it is the enzyme of choice for these applications. The lack of 3'→5' exonuclease prevents the labeled nucleotides from being removed after they are incorporated by the polymerase. The enzyme is used in the NEBlot Kit (NEB# N1500) for making high specific activity probes using random primers. It is also used in the NEBlot Phototope Kit (NEB# N7550), which generates biotinylated probes.
Q7: Are NEB DNA Polymerases supplied with dNTPs?
A7: 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.
Q8: Are there other methods for making probes?
A8: T7 RNA Polymerase (NEB# M0251) and SP6 RNA Polymerase (NEB# M02070) and the HiScribe RNAi Transcription Kit (NEB# E2000) can be used to make RNA probes. Increased levels of detection in nucleic acid hybridization can be obtained due to the greater stability of RNA: DNA hybrids with respect to DNA: DNA hybrids (1).
(1) Zinn, K et al. (1983) Cell 34, 865-879.
Q9: What is the enzyme of choice for chewing back 3' overhangs and filling in 5' overhangs (3' recessed ends)?
A9: DNA Polymerase I, Large (Klenow) Fragment (NEB #M0210) 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.
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