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TOPO TA pCR 2.1 cloning - (Mar/09/2005 )

Hi all,

recently I used TOPO TA cloning kit to clone a piece of gene with ~900bp. I transformed the plasmid into E.coli DH 5รก and then had blue-white screening. After around 4 weeks, I picked ten white clones to do PCR with M13 primers. There are some clones produced positive result where insert size was correct. But those PCR with negative clone did not even show up a band of 201kb, there was nothing except some smearing on the gel (even without insert, the cells which grow on LB+AMP plate should produce a band of 201kb in PCR since I use M13 primers). So, I checked on my plate, whether they contained functional antibiotics. But my plate was okay. and then I check on my PCR reaction, is okay also. I felt a bit bizarre on what is happening in my experiment.

Which steps I can check further?

Thanks a lot!

Ming Woei


If you got positives why do you care about the negatives? Just go on with the positives. There is always some weird stuff happening in molecular biology so unless you are studying molecular biology just be happy you got positives! smile.gif

PS. I think you mean you should get a band of 201bp not 201kb.