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


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. 
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,
David




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