Cloning problem using pET32a vector
Posted 24 January 2012 - 09:00 AM
Transformation of uncut pET32a gives a lawn of bacteria on LB + Amp agar (vector is OK)
2hr, 4hr, and overnight NdeI and EcoRI digests give the same results - no colonies
After each digest, and pfu fill-in both vector and insert are purified by Qiaquick gel extraction
I have tried a 1:3 and 1:8 vector to insert ratio
I have tried both NEB Quick ligase and Promega T4 DNA ligase
One tricky part is that it is not possible to tell if the ecoRI digestion was successful by gel visualization since very few bp are lost in the digestion. Also, I lose much of my vector after pfu fill-in and by the end of all of the digests and purification I am only left with about 40-50 ng of vector.
Any advise on this problem would be greatly appreciated. I am leaning towards the problem being not enough vector and I am not sure on how to go about increasing my yield. Also, I get a bit of a smear/double band after I digest pET32a with NdeI even overnight which might indicate incomplete digestion.
Posted 24 January 2012 - 11:53 AM
Have you considered using PCR to amplify your vector, adding whatever enzyme sites you want to the (now linear) cloning site?
I would check the UV exposure of my gels, which can often dramatically reduce transformation efficiency.
Posted 24 January 2012 - 01:22 PM
Posted 24 January 2012 - 04:46 PM