I cant get colonyrace cloning
Posted 13 September 2011 - 09:44 AM
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
Posted 13 September 2011 - 01:15 PM
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.
Posted 13 September 2011 - 06:20 PM
Posted 18 September 2011 - 08:50 PM
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.