Maximum plasmid insert size?
gal-1 at bones
Sat May 13 09:56:17 EST 1995
On 12 May 1995 HAVILAND at KIDS.WUSTL.EDU wrote:
> 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.
Usually the ligation efficiency of large inserts is
not the main issue. The main problem is getting a large piece of DNA
bacteria once the ligation has been accomplished. Usually plasmid
subcloning shows a dropoff around 10-15 kb because Ca++ shock
transformation seems to have some size dependence. I have subcloned 15
kb fragments in Bluescript, but as noted above, it was difficult.
However, having talked to a number of people who routinely handle very
large plasmids, I got the message that electroporation will handle much
bigger plasmids (20-40 kb).
My suggestion would be to try electroporation to transform your bacteria,
and also, as noted above, to prescreen by hybridization or blue/white
selection to cut down on your background.
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