Blund ends cloning

Fiberhead stern100 at mail.concentric.net
Mon Jan 6 16:54:13 EST 1997


>Greetings everyone,
>
>I am trying to clone a Vent DNA polymerase amplified PCR product in to a 
>vector which was cutted blunt by restriction enzymes.
>
>It didn't work, of course. Since the primers used for PCR were not 
>phosphorylated at 5' ends. Instead, the restriction analysis of the 
>transformed colonies showed that the vector ligated to themselves.
>
>So, my question is, first, whether anyone has some strategy to clone 
>blunt end insert with 5'OH successfully. 

That strategy requires very high concentrations of insert to actually
suppress the background vector religation.  It can be done with synthetic
oligos, but it would probably be nearly impossible with a PCR fragment.

>Second, if the insert has to be phosphorylated at the 5' ends by T4 
>polynucleotide kinase, is that ok to use T4 DNA ligase buffer to do it. 
>The reason I am asking is that it is holiday season now, what I have is 
>only the T4 kinase but not the buffer. I found the T4 DNA ligase buffer 
>(Gibco/BRL) is very close, except there are 5% PEG-8000 and use DTT 
>instead of ME.

Dude, why dont you just make some PNK buffer?  And try kinasing the oligos
before PCR , it will probably be more efficient.

-seth





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