Problem cloning PCR product into pCR-Script

Koen De Smet k.desmet at
Wed Mar 11 10:21:20 EST 1998

We routinely use pCR-Script from Stratagene to clone our PCR products. 
The PCR products are first run on a gel, cut out and then cleaned up 
using the QIAEX-II kit from QIAGEN. An aliquot is then used to ligate in 
the vector, according to Stratagene's protocol (except that we do not 
quantitate our insert).

After transformation we get around 50% white colonies on IPTG/X-gal 
plates; but we have a similar ratio on a vector-only control plate. When 
we pick colonies for minipreps (from the plate that should have clones 
with our insert), some of the white ones are pCR-Script with our insert, 
but the majority have what looks like vector only with deletions. The 
vector is 2.9 kb, but these clones have single bands ranging in size 
from 1.5 kb to around 2.9 kb. 

We shouldn't get such a high number of false negative clones. Can 
anybody explain this?

Koen De Smet
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     Imperial College School of Medicine at St Mary's									

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