subcloning problems and need help..please!!

newsnet customer customer at newsnet.com
Thu May 4 05:34:24 EST 2006


> hi
>
> i am trying to subclone a target dna from pskt to plox. the target dna has
a gene of interest-linker-gfp. the restriction digestion of the pskt vector
releases a correct >size of 2.7kb. but after inserting the fragment in plox,
we see the linker missing??  on sequencing we see the sequence of gfp and
the gene of interest but no linker. >the linker is a small fragment of 20-30
kb. the subcloning done in XL10 and XL 1 bacteria.

20-30 kb is not a small fragment. I assume you meant 20-30bp. If so, the
linker would be too small to resolve the difference between GOI-linker-GFP
and GOI-GFP on an agarose gel.

> please let me know as to how to over come the problem.

Two things come to mind: linker is absent from your vector OR linker is cut
out from restriction digest.

> thanx a lot..
>
> Deb

ST










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