I cant get colony - (Sep/13/2011 )
I have a trouble and I hope you can help me. I am using RACE technic. I got positive result after pcr reaction. Some samples gave 2-3 band, so I made jel prufication.Then I run my sample and they gave very light bands. I made ligation (TOPO vector invitrogen) and cloning. But I couldnt obtain colony, actually I got only 1 blue colony no white colony. I repeat my protocol and same results. What do you think about problem.
And more question ıf I have only 1 band, can I sent it to sequensing directly
And ıf I have only 1 band can I get cloning directly without jel prufication
Which polymerase are you using to amplify your products?
You can sequence a single band PCR.
With a single band PCR you can proceed directly to cloning without gel purification, though you should still clean up the PCR product.
I used clontec kit It has polymeras. But my kit isnt new. Maybe it is problem. Actually this is my 2. race. I got partial sequences. So I redesign primers and did again reaction My vector and plasmid are new.
What polymerase are you using? When TA cloning, your PCR product must add an adenine at the 3' end of your product in order for it to ligate to the vector. By my understanding, proofreading enzyme will not add the A to the end of the product-check if your polymerase will. You can add the A after the gel purification using Taq/nucleotides, etc... google for a protocol.
I've done heaps of TA cloning using the TOPO pCRII Invitrogen kit, after PCRs using Taq, followed by gel purification, and it's usually quite easy.