Sub cloning

L.D. Ofner BMBLDO at leeds.ac.uk
Fri Aug 1 08:28:01 EST 1997


Hi everybody,

I've been trying to sub clone a full length cDNA sequence (4.5kb) into an 
expression vector for ages now with no luck and it's driving me mad ! There 
are EcoR1 sites at either end of the insert in its original vector which I'm 
using to cut it out (there are no other restriction enzymes that I can use 
for directional sub cloning which won't cut the insert sequence or the 
expression vector in the wrong place). I've performed the necessary 
restriction digest for cutting the insert out and cutting the expression 
vector, purified both from agarose gel using Wizard clean up and 
dephosphorylated the expression vector ready for ligation. I've set up 
numerous ligations with differing ratios of insert:vector (including having 
insert in vast excess) and have had loads of colonies growing on plates. But 
when I prep up the DNA ( usually pick at least 10 colonies) and check by 
restriction digest, none seem to contain insert ! I've checked that the 
vector is dephosphorylated by tranforming my competent cells with it and I 
get no resulting colonies on plates. Help ! Where am I going wrong? I'd be 
grateful for any advice or suggestions (soon before I go totally insane !).

Thanks. Lisa. 



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