Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

TA Cloning of alpha-1,3 glucanase: No blue colonies


  • Please log in to reply
5 replies to this topic

#1 sfora71

sfora71

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 19 March 2013 - 10:36 AM

Hi, my lab group is studying an organism with 4 genes coding for alpha-1,3 glucanase. We isolated the genes, extracted RNA, and amplified the genes by PCR with 2 cDNA pools. We know our PCR was successful (gel electrophoresis: band sizes were correct).

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.

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,793 posts
406
Excellent

Posted 19 March 2013 - 04:47 PM

Are you sure X-gal and IPTG were added to the plates? For TA cloning a week may be too much, they are definitely better fresh. Did you use Taq for the amplification?

#3 sfora71

sfora71

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 19 March 2013 - 06:54 PM

The student adviser prepared the plates and says they definitely had X-gal and ampicillin on them, but who knows if that's true. The manual from Invitrogen says to use IPTG only if TOP10F´ cells were used, but the adviser never told us which strain of E. coli were added to the plates. I don't recall her saying anything about using IPTG, but I could've missed that. If that was a requirement for whichever strain of E. coli she used and it was left out, how would that affect the results?

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.

#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,793 posts
406
Excellent

Posted 20 March 2013 - 12:14 AM

IPTG isn't strictly necessary, but it can help. Don't worry if it wasn't there.

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.

#5 sfora71

sfora71

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 08 April 2013 - 02:31 PM

Ok, so after several unsuccessful runs, our RD realized she didn't program the thermal cycler correctly. So the annealing temperature was set at 50°C instead of 55°C and the elongation period was set for 1 minute instead of 2.

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.

#6 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,793 posts
406
Excellent

Posted 09 April 2013 - 12:50 PM

A lower annealing temp may affect the PCR in that you will usually get lots of non-specific bands, but it can also result in a complete failure of the PCR (usually due to primer heterodimerisation). The absence of clones when you have used a PCR product which you know has bands, indicates that the cloning has failed for some reason. Ligations are notoriously tricky and can be affected by a whole raft of different things, but the most common problem is too much salt in the ligation. I would need to know a bit more specifically how you purified the PCR product before I can say if there may have been a problem there.

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).




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.