trouble in TOPO TA sequencing (View forum version)



karthikmgt

Posted 16 October 2010 - 12:24 AM

hi budding scientists,

I am trying to clone and sequence a 428bp pcr fragment (taq pol amplified) in pCR4 TOPO vector... the TOP10 competent cells provided by the kit manufacturers are out dated so i used DH5alpha cells(self made)...... i followed kit protocol for ligation.. and since it is my own comp cells i did heat shock for 2 min at 42C and plated cells and found no clones the next day.... this is the background of my prob....
now my question is... the pCR4 TOPO vector has lac promoter and i did not use IPTG or X gal because the vector has a bacterial lethal gene fused to the lacZ alpha part... so according to the kit manufacturers one can see clones only if they carried insert containing plasmid.. the religated ones will be killed....

now taking all this into consideration... should i use IPTG to activate the plac in the vector so as to get clones....

in a nut shell i wanna know whether i can use dh5alpha and pCR4 TOPO vector to clone my fragment... if yes should i use IPTG?

any suggestions are welcome......

phage434

Posted 16 October 2010 - 05:26 PM

I'll bet your problem is low efficiency competent cells. Your first step should be to measure the competence of your cells. Transform 10 pg of a plasmid such as pUC19 (do a serial dilution of a stock of known concentration). You should get 100's of colonies. If you get 1-2, you have a problem. If you get none, you have a serious problem. Two minutes sounds like too long a heat shock -- we use around 45 seconds, but that is probably not the problem.