CaSpeR cloning - Help sought
westb at VAX.CS.HSCSYR.EDU
Fri Jul 1 14:48:24 EST 1994
The only thing I can tell you after many years at this is that big
vectors are difficult to clone into. For example, yeast vectors are the
same size as Drosphophila ones (9 to 12 kb) and the effect is the same.
Don't try blunt-end ligations whenever possible, stay with sticky end
cloning. Also, keep the cloning simple at the point you're placing the
fragment into the fly vector- do all complicated manipulations in pBS etc.
Finally, when all else fails the motto we've always lived by: " SCALE UP!".
That is, ligate 1 to 5 ug of insert and vector instead of ng amounts.
That probably doesn't help, but at least allows me to say hi. Hope
all else is going well there.
At 6:01 PM 7/1/94 +0000, jonesc wrote:
>A colleague and I have been struggling to subclone various genomic
>fragments (ca. 8-12 kb) into pCaSpeR4 for plasmid rescue experiments,
>but have been having the devil's own time getting recombinant plasmids.
>Does anyone have any suggestions beyond using massive amounts of insert
>and vector (which we've been doing) and Herculean effort (ditto)? Is
>there something peculiar/tricky about this vector, or large vectors, or
>large inserts? I've tried looking around in what seem to me the right
>places, but have come up empty-handed. Thanks for any help you have to
>Chris Jones (jonesc at cshl.org)
Department of Biochemistry
and Molecular Biology
SUNY Health Science Center
750 E. Adams St.
Syracuse, NY 13210
westb at vax.cs.hscsyr.edu
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