I want to insert a 1k insert into a 13K plasmid.
I amplified the insert with primers that added an xba1 site to the 5' end and a kpn1 site to the 3' end, and got a nice strong band after PCR. I cleaned this up with a Qiagen PCR cleanup kit, and then performed the double digest. Kpn1 can't be heat inactivated, so I used the PCR cleanup kit to purify again.
I digested the plasmid with the same two enzymes, and ran some on a gel alongside uncut vector to confirm that it's been cut. Since it's too big for the Qia kit I cleaned it up this reaction by phenol chloroform followed by spin-dialysis. I checked on a gel and still have a decent concentration of cut vector.
I tried to spec check, but got some odd readings for the vector - much too high, especially considering the moderate strength band I observed on the gel.
Because of this, in a 10ul ligation reaction with t4 ligase, I simply used 1ul of vector and three of insert. I let it ligate for 4 hours, then transformed 4ul into XL1 blue and DH5alpha electrocompetent cells (since I sometimes get better results with one or the other) and plated on selective plates. No colonies.
Any ideas where I should start to work out what's gone wrong?
Edited by Scuffle, 02 July 2010 - 01:44 AM.