I am trying to make blunt ends out of nicked plasmid DNA using random hexamers and Klenow polymerase (large fragment) from NEB which has 3'-5' exo but no 5'-3' exonuclease activity. My goal later is to use this blunt ended DNA for Adaptor/Linker ligations. Unfortunately, in the method I am using, I can use only Blunt ends, not sticky ones. To test it, if I take a blunt ended PCR amplicon, it ligates well, but if the PCR amplicon is denatured with random hexamers and quick chilled, blunted with Klenow, the ligation never work. I used this test when I failed to ligate anything to the Klenow blunted nicked plasmid. I have tried different protocols for Klenow (incubation temperatures etc) and different methods of purifying DNA after Klenow, (columns, Phenol chlorofom, heat inactivation etc). Nothing seems to work. Can anybody suggest what might be the problem and any solutions? Thanks
Hi Sanjay, I really can't understand what you are trying to do.
Anyway, the Klenow is normally better working to fill in 5' overhangs, it is not the best way to blunt 3' overhangs, because similarly to most Taq polymerases (except the Pfu) tends to add extra nucleotide overhangs at the 3'.
A good blunting enzyme, still from NEB, is the Mung Bean nuclease that acts both on 3' and 5' overhangs.
Hi Pietro, the design of the experiment is really complex which I will not be able to explain in less than a page.
But the klenow I am using is not exo-, it still has 3'-5' exo activity according to NEB. So ideally it should work. But your idea if good too, I will try Mung bean nuclease. I also have Pfu turbo, I will it a try with this too. Thanks,
Sanjay
If you use a typical Taq polymerase, you can use a TA cloning kit.
I don’t understand your experiment. Why do you need to clone random extension products? If you denature DNA and then add Klenow and random hexamers you will get random priming with different product sizes whose sequences might be very complex to predict..... You only get extension products no amplification.
Anyway, as suggested by Premi you can add Mung bean nuclease which is specific to ss DNA.
Hi Abdelhalim Boukaba,
I am actually looking for these random products. The experiment is to have single stranded nicks at specific points using a system and then identify these points by deep sequencing using adapters ligated to the places where there were nicks. In order to do that I will have to denature DNA and make it blunt ended at the nicks using Klenow, or other polymerase. Mung bean will only chew single stranded extensions which is of no use for my experiment. Also, one of the adapters is asymmetric, so mung bean will destroy the extension and I will concatemers.
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