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Blunting Ends with Klenow and T4 DNA Polymerase

Klenow Repairs 5' Overhangs to Generate Blunt Ends

  • 5'ATGCATGCTACG
  • 3'TACGTACGTACG
  • 5'ATGCATGC ATGC
  • 3'TACGTACG
  • Fill-in recessed 3' termini
  • 3'=>5' exonuclease
  •  
  • Very weak -- Use T4 DNA polymerase
  1. In a 20-µl reaction, digest 0.1 to 4 µg DNA with reaction enzyme.
  2. Add 1 µl of 0.5mM dNTP. It is unnecessary to inactivate the restriction endonuclease, to change buffers or to repurify the DNA prior to adding the Klenow fragment.
  3. Add 1 to 5 units of the Klenow fragment and incubate at 30°C for 15 minutes.
  4. Stop the reaction by heating to 75°C for 10 minutes or by adding 1 µl of 0.5M EDTA.

For restriction fragments produced by cleavage with different endonucleases, it is possible to repair one end selectively. This is done by cleaving with enzyme 1, repairing the ends, inactivating the Klenow fragment by heat (75°C for 10 minutes), and cleaving with enzyme 2.

T4 DNA Polymerase Trims/Fills in 3' Overhangs

This protocol employs the activity of T4 DNA polymerase, which catalyzes 3'=>5' synthesis from primed single-stranded DNA. The enzyme has a 3'=>5' exonuclease activity, but lacks 5'=>3' exonuclease activity.

  • 5'ATGCATGCTACG
  • 3'TACGTACGTACG
  • 5'ATGCATGC ATGC
  • 3'TACGTACG
  • Fill-in recessed 5' termini
  •  
  • 3'=>5' exonuclease
  •  

For fill-in, set up the same and incubate at 12-16°C for 15 minutes.

Note: At higher temperatures the exonuclease activity is very active.

Protocol 1: Blunting DNA with with T4 DNA Polymerase

Using digested DNA which has been precipitated and resuspended in water...

  1. Prepare the 5X Reaction Buffer*
  2. Incubate the mixture at 11°C for 20 minutes, or at room temperature for 5 minutes.
  3. Stop the reaction by adding EDTA to 10mM final concentration, then heat at 70°C for 10 minutes.
  4. Extract with phenol:chloroform and precipitate DNA for further use.

*5X Reaction Buffer

  • Digested DNA
  • 2mM dNTP Mix
  • T4 DNA Polymerase
  • Water, Nuclease-Free

4 µl

  • 1 µg
  • 1 µl
    (0.1mM final concentration)
  • 1 unit
  • Q to 20 µl

Protocol 2: T4 fill-in with larger amounts of DNA

Using digested DNA which has been precipitated and resuspended in ddH2O to a final concentration of approximately 500 µg/ml.

  1. Set up the following reaction(s)**.
  2. Stop the reaction by adding EDTA to 10mM final concentration, then heat at 70°C for 10 minutes.
  3. Extract with phenol:chloroform and precipitate DNA for further use.

**Reaction Mixtures

Small Reaction (1 to 10 µg DNA)

Final reaction volume is 50 µl.

  • 5 µl of 10X T4 DNA Polymerase Buffer
  • 2.5 µl 2mM dNTPs
  • 2.5 µl 2 mg/ml BSA
  • 2 to 20 µl DNA (1 to 10 µg of DNA)
  • 5 units T4 DNA Polymerase
  • 20 to 38 µl ddH2O

Large Reaction (10 to 30 µg)

Final reaction volume is 100 µl.

  • 10 µl 10X T4 DNA Polymerase Buffer
  • 5 µl 2mM dNTPs
  • 5 µl 2 mg/ml BSA
  • 20 to 60 µl DNA (10 to 30 µg of DNA)
  • 10 units T4 DNA Polymerase
  • 20 to 60 ddH2O
  • Incubate at 12°C for 20 minutes or 37°C for 5 minutes
  • Inactivate at 65°C for 10 minutes

© 2014 The University of Texas MD Anderson Cancer Center