Primers annealing protocol

brett brett at BORCIM.WUSTL.EDU
Wed Apr 9 11:30:18 EST 1997


>>Pascal Mertens wrote:
>>> 
>>> Hi there,
>>> 
>>> WE want to anneal two primers (135 bases lenght) and to clone them in a
>>>plasmid.
>>> Here is our first assay protocol:
>>> We mixed 5nmol of each oligo (in TE buffer) to a final volume of 111 microL.
>>> The mix was heated for 5 min at 95 degrees, and then cooled down to 45 (1
>>> degree per min) and then transfered on ice.
>>> 
>>> We didn't obtain any ligation product. We analyzed the annealing product
>>> on acrylamide gel: there was a band at the same level than for the oligos
>>> (ran separately) and a light smear upper to this band. So the annealing
>>> does not seem to be good.
>>> Any advices?
>Hi!
>
>I use 1xM buffer for the annealing (M restriction buffer from
>Boehringer). Heat it up to 95-100 C for 5 min, then let it slowly cool
>down.
>In the ligation I use 100 times more oligo ends than vector ends.
>
>Good luck!
>
>Csaba 
>csakis at ki.se

I agree that your choice of buffer was unfortunate. I recommend a Mg++
containing buffer, such as most any RE buffer, which will allow duplex
formation. Good luck!


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             




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