cloning

Klaus Salger salger at wap18.zi.biologie.uni-muenchen.de
Mon May 5 19:55:08 EST 1997


Els Van Geldre (Els.VanGeldre at rug.ac.be) wrote:
: Hi,

: I'm trying to clone a PCR product in M13mp18 RF. After cutting the 
: vector with Sma I and and blunting the 5' side of my PCR product I obtain 
: few bleu plaques (I treated the vector in order to minimize selfligation) 
: and much more white plaques. However, when I try to isolate ssDNA and 
: sequence the insert with a universal primer on the M13 vector I 
: notice that there is absolutely no insert present. I repeated the 
: experiment several times, what goes wrong ?
: Maybe, the problem is correlated with the fact I purify vector and PCR 
: product with the Geneclean kit from Bio 101 ?
: Thank you for answering,
: Els

Els,

I guess the problem is that SmaI is able to nibble back the ends of the cut
DNA. This problem  has been reported repeatedly in this newsgroup. So, if
SmaI damaged the ends of your vector you will end up with white colonies
without inserts because the lacZ-fragment has been inactivated (eg by a frame
shift). If this is the case you should see it in the sequence!
 
You'll find the old postings in the archive on WWW.BIO.NET.

Cheers
  Klaus

--
Klaus Salger		phone : +49 (0)89 5902 	-502
Zoologisches Institut	FAX   :			-450
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