Maximum plasmid insert size?

Warren Gallin 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
> 
> 
> Uwe:
> 
> 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 
into to 
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.

Warren Gallin



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