TA Cloning of alpha-1,3 glucanase: No blue colonies
Posted 19 March 2013 - 10:36 AM
We ligated with Invitrogen's TA Cloning Kit with pCR 2.1 and incubated overnight. Next we attempted to transform the ligation into competent E. coli. When analyzing the colonies, we noticed that on the plates for three of the genes, there were lawns of only white colonies. For the fourth gene, there was no growth whatsoever.
What does this mean? Obviously there was a mistake somewhere. The only thing I could see that might be problematic is that our PCR products were a week old, but that doesn't explain what happened on the fourth plate (which definitely had ampicillin on it). Our student advisor is stumped too. This is a lower level course, so I have no experience with these procedures and the upperclassman isn't great at explaining things.
Posted 19 March 2013 - 04:47 PM
Posted 19 March 2013 - 06:54 PM
Each student in our group was responsible for preparing PCR tubes for one of the four primer sets using 2 different cDNA pools. The master mix we used was supposed to have Taq polymerase in it, but we were not responsible for preparing the master mix, so once again I can't be sure it was used.
Posted 20 March 2013 - 12:14 AM
If you had products on the gel from the PCR, a polymerase was used. I was checking that it was taq as it adds an A overhang to the PCR, many other polymerases don't do this. Try adding some PCR product to mastermix and incubating at 72 for 3 min, then do the cloning again straight away.
Posted 08 April 2013 - 02:31 PM
We did transformations again and got blue and white colonies on 4 of the 8 plates (from chromosomes 3 and 14). But I need to understand why we didn't have successful transformations for the others, and why we had such weird results the first time around. And unfortunately, no one has been able to offer any guidance on the subject.
Posted 09 April 2013 - 12:50 PM
A complete absence of clones might indicate that you have a toxic gene product or that for some reason the ligation reaction has completely inhibited the transformation (quite unlikely, I would expect a few colonies at the least).