Subcloning problem

Luis M. Alvarez luism at helix.nih.gov
Mon Aug 21 17:47:22 EST 1995


I've been trying to clone a small DNA fragment (around 200 bp) into the 
pOPI3 and pOPRSV vectors from the Stratagene's LacSwitch kit. These 
vectors contain the B-lactamase gene, ColE1, Neo gene, f1 origin, RSV LTR 
and SV40 intron and poly A for correct procesing. The size of the vectors 
is around 6 Kb. Now, these vectors only contain a Not1 site for cloning. 
So, I PCR-amplified my fragment with oligos containing Not1 sites in an 
appropiate configuration for good Not1 digestion.
The problem is that after ligation/transformation something happens that 
delete the vector to a 3Kb size with no insert (?). I have try different 
E. coli hosts, different insert sizes and different vector preparations 
and the thing keep doing the same. What is even worst is that the vector 
alone is pretty stable in all hosts and the insert is clonable in other 
vectors. Nobody around here seems to know what is happening.
Any suggestions?

Thanks,

Luis Alvarez
luism at helix.nih.gov



More information about the Methods mailing list