PCR Question

Huseyin Kucuktas kucukhu at mail.auburn.edu
Wed Jun 28 16:02:41 EST 1995


	
	Hello everybody,

	I am trying to amplify a 4.3 kb fragment from a virus to subclone 
into a common vector. I have designed primers (one is a 27 mer with a 
BamH1 site, other one is a 31 mer with a EcoR1 site. For efficient 
cutting I had to add 2 bases to the ends) that are complementary to the 
5' and 3' ends of the ORF. Using enzymes that can amplify up to 20 kb 
fragments I amplified the fragment that I needed. However, I could not 
clone it at all. Here are the things that I have tried so far:

	- Directly digested the PCR product with both enzymes at the same 
time using compatible buffer (BRL's React3), gel purified the fragment 
(with geneclean) and try to clone into pUC19 treated same as above.

	- First gel purified, genecleaned and then digested the fragment same as 
above and try to clone it.

	- Tried to blunt end the fragment using T4 DNA pol and clone into 
Sma1 cut pUC19. I did the same thing with using Klenow also.

I use DH5alfa electrocompetent cells, my linear pUC controls work, but I 
don't have any colony on the plates. (Sometimed 2-5 blue colonies).
	
I would appreciate any comment..



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