subcloning pcr bands
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.
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
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)
1660 S Columbian Way South
Seattle WA 98108
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