Large-ish vectors that won't behave

bcraft at darwin.bio.uci.edu bcraft at darwin.bio.uci.edu
Tue Nov 22 23:21:04 EST 1994


I am doing some cloning  with vectors for use with the yeast two-hybrid 
system.  I am using pMA424 (approx. 10kb) and pGAD2F (approx. 12 kb).  The 
problem I am having is with ligation and transformation efficiency.  When I 
transform the pMA424 plasmid in its supercoiled state I only get an efficiency 
around 10e5 as opposed to an efficiency of about 10e7 with pGEM.

For my ligations I have tried to use 0.1ug of vector and ratios of 1:1 and 
1:10 vector:insert.  I have succeeded in ligating in a 22bp insert like this, 
but only had 6 total colonies under these conditions (3 of which had vector 
and insert, 2 of which had only one insert).  Anyway, I am now trying to 
ligate into the same vector (pMA424 + 22mer) a 750bp insert without any 
success using the above amounts of DNA and ratios of vector to insert.  Can 
anyone give me any advice for using these large vectors (my fellow labmates do 
not have problems, but they use much smaller vectors)?

Any insights at all?

I am using E. coli strain HB101 for my transformations.  Also, I use Qia Preps 
for my mini preps which has been giving me very clean DNA.

Thanks

Brian Craft
bcraft at darwin.bio.uci.edu



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