missing vector part

chrissyl at my-deja.com chrissyl at my-deja.com
Wed Aug 25 23:21:31 EST 1999


I cloned some of my inserts into pGEM-T vector and after transformation
process I PCR screened the positive colony (using M13 F and R) and
obtained a band with a slightly higher size than the original insert
(1800bp insert indicated by the screening). With the positive clones
(although low in numbers) I did an overnight culture using 2xYT medium
and did a plasmid prep. When I did a restriction digest to these clones
only some of the digests worked successfully (that is restrictions using
the enzymes which cuts downstream from the insert), the other digests
that didn't work involve the R-enzymes whose sites are upstream from the
insert. And those resctriction that did work showed a band with size
3000bp (the size of the whole vector), instead of 4800bp. Hence this
indicated the absence of inserts. When I sequenced the clones, I found
out from the sequence that one section of my vector is missing (which
include some of the restrciction sites).

So if anyone can actually help me out in the reasoning of this result
would be very appreciated. One suspicion is 'plasmid rearrangement'
which seems far fetched, but possible.

Please let me know if you could answer my problem or have experienced
some thing like this. Thanks.


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