Digest, refill an digestion - How can I do it? (Jun/15/2005 )

I have two plasmisds: Pires-GALNS and AATp.

From Pires-GALNS I remove CMV promoter with 5'NruI(blut ends)-NheI(cohesive ends)3'. From AATp I remove promoter AAT region with 5'BglII-EcorI3'. All that ends are not compatible and for that reason I use Klenow pol in order to refill and become all in blut ends. Then I use T4 ligase to ligate AAT promoter with Pires-GALNS without CMV promoter.

After ligation, I transform competent E.coli Ultragold. However, I have not gotten clones. Someone could help... really I have used many different protocols.

thanks for your help

My first suspect with be the Klenow is not working. Try T4 DNA polymerase, keep you dNTP's in molar excess and run the rxn for 5 min then inactivate the enzyme.
Promega's is pretty good and the rxn works for me.