Posted 22 June 2010 - 09:11 AM
I've been trying to ligate ampicillin resistance into a vector and I'm not sure what I'm doing wrong. I've digested both, dephosphorylated the vector, PCR'd the plasmid with the desired casette, digest both (though accidentally together but then I ran them on a gel and that seemed to separate them). I've ran controls that lead me to believe it's a transformation issue (I made dephos. Ligase + and ligase - plates with no results on either, I made digested vector ligase + and ligase - plates with no results, and I also did a test on my transformation efficiency because I was incubating in a heat block as opposed to a shaker but my TE is still low even in the shaker with the parent vector). I CAN transform, but apparently with low TE.
During transformation I incubate on ice for 30 minutes, heat shock at 42C for 2 minutes, ice for 2 minutes, recover fat 37C for 60 minutes.
I'm pretty lost and any suggestions/help would be appreciated.
Posted 22 June 2010 - 09:30 AM
And what do you mean with digest both?
you did an digest on the gene too? If there is a restriction site in the gene then your gene is ruined.
(but you say the gels of both are ok?)
You might also wanne try an electrotransformation.
Posted 22 June 2010 - 09:54 AM
I think I also may have not been as gentle as I needed to be with the cells, but even with that I was able to clone the parent vector. I'm going to try a shorter heat shock duration on transformation of the parent vector to test if that increases my TE. I'd like to get this finally done though so that I can move on.
I'll also do the extra ethanol precipitation step like you said though I did centrifuge the minicolumn assembly in order to evaporate the ethanol
Posted 24 June 2010 - 09:40 AM
Anyway: try to digest them seperately (so you can put them on different gells or different lanes at least). And I would also try an electrotransformation.
And you are sure you used the correct restriction enzymes?