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
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