Unstable Construct?

Lyndal Kerr lyndal at deakin.edu.au
Wed Jun 27 19:02:12 EST 2001


I am trying to subclone a gene into pGEX4T for some future expression 
work.  My problem is as follows -

Exp. 1 - Transformed ligation mix into DH5alpha cells, colonies grew 
well.  Checked positive clones by colony PCR.  (In our lab this involves 
picking the colony, spotting onto a master plate, and mixing in the PCR 
reaction mix.  Master plate is incubated at 37 degrees O/N).  Colony PCR 
revealed that 7 of the 8 clones were positive.  1 colony contained 
re-ligated vector only - this was the only colony that grew on the master 
plate.

Exp. 2 - Transformed the same ligation mix directly into the expression 
strain BL21.  Yielded two distinct type of colonies, normal well rounded 
colonies (all tested contained re-ligated vector) and much smaller colonies 
(all tested contained the gene of interest).  On the colony PCR master 
plate only clones that contained the re-ligated vector grew.

Exp. 3 - Tried DH10B cells as in Exp. 2.  Results identical!

My only option at this point is to try and grow the clones in liquid 
culture directly from the original transformation plate......but I am not 
sure this will work.  If anyone has any suggestions or has encountered this 
problem in the past, I would gladly appreciate the help!

Thanks in advance - Lyndal

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