PCR Question

Christopher M. Tebeau ctebeau at melre.acns.nwu.edu
Tue Jul 4 09:52:51 EST 1995


In article <Pine.SOL.3.91.950628154527.9695A-100000 at lab1>, 
kucukhu at mail.auburn.edu says...
>
>        
>        Hello everybody,
>
>        I am trying to amplify a 4.3 kb fragment from a virus to 
subclone 
>into a common vector.....

  I had a similar problem recently trying to subclone a 1.5 kb phage 
fragment using primers with pstI on each end.  I got it to clone as 
follows:  gel purify pcr reaction, digest entire elution, phenol 
chloroform extract and etoh precipitate the digest, ligate 1/10 and 
9/10 of digest O.N. etc...  When I did this w/o the phenol:chloroform 
step it didn't work.  I am not sure why??  
  I have also subcloned smaller pcr fragments 150-500 bp with BamH1/EcoR1 
primers with the same steps.  Here I thought it would be important to 
inactivate BamH1 by P:C before the ligation because it doesn't heat 
inactivate according to the Gibco catalog.  Anyway, I hope this helps, 
and if anyone knows of a good discussion in the literature about cloning 
PCR products I would appreciate hearing about it.

					Christopher M. Tebeau
					Northwestern University
					ctebeau at merle.acns.nwu.edu 




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