Maximum plasmid insert size?
David L. Haviland, Ph.D.
HAVILAND at KIDS.WUSTL.EDU
Fri May 12 14:41:34 EST 1995
In <tiedtke-0505950937570001 at pbio16.uni-muenster.de> tiedtke at vnwz00.uni-muenster.de writes:
> Dear Netters,
> what is the maximun insert size for plasmids like pUC18/pUC19 and pT7T3?
> Uwe Baumert
I have experience with Bluescript and pSP72. Most everything that I've
tried to ligate into these two vectors has gone right in. In these cases,
the insert sizes have ranged from a few hundred to upwards of 6-7 kb.
Beyond that, inserts (for me at least) have been more problematic to get
into a vector. Overall, I've noticed a decrease in the number of
recombinants over background with inserts up to 10 kb. The one time I
"had" to get something big (14 kb) into Bluescript it took me three
separate ligation attempts to get anything over background that was worth
screening. The third-time was a charm as the ligation was barely above
background and I opted to do lifts and probe with the insert (14kb), rather
than too many mini-preps. I had 1 colony on the entire 3 ligation plates.
Fortunately, that was all I needed.
I'm sure someone out here in net-land has ligated even a bigger insert but
14 kb is my personal best. Given the amount of work, if I had it all to do
over again, I'd probably would have tried to sub-cut the isolated band and
put it into two separate vectors instead.
Hope this helps,
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