subcloning pcr bands

L. Bastian yacman at homer17
Sun Aug 4 09:52:11 EST 1996


Enrique Graziano-Areste (egagmp at ac.upc.es) wrote:

: Hello everybody,


: 		I am trying to clone some PCR bands from a 5' RACE.
: The main problem I have is that only one out of 70 white colonies
: use to have the insert, the other ones are white, but when I make
: PCR using the forward and reverse primer over the colony I get 
: only a band that is the polylinker (about 200 bp using pBLUESCRIPT).
: How can I increase the efficiency of my subcloning procedures?. If
: you need more details please do not hesitate in asking me. Please,
: help me. I am starting to think about suicide...
: 		Thank you in advance.



: 		Enrique

I don't use blue-white selection.  I have seen too many empty white 
colonies and too many blue colonies with inserts, some as large as 1kb.  I 
think it works sometimes, but often its just a lousy screen.  If you are 
considering suicide, you might consider the pZero vectors that someone 
else suggested, which sound like a good idea although I have never tried 
them. :)

Good luck

--
L. Scot Bastian Ph.D.			206-762-1010 ext. 1918 (voice)
University of Washington at the         206-764-2689 (FAX)
  Veterans' Affairs Medical Center      yacman at u.washington.edu (Email)
M/S 151 
1660 S Columbian Way South 
Seattle WA 98108 




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