Problem with TA PCR cloning - no insert in white colonies - (Mar/22/2005 )
I haven't read the manual but it seems strange that the degradation of the T overhang can produce white colonies but insertion of short fragments of DNA will produce blue colonies. I could understand if the fragments were in multiples of three and therefore kept the beta-galactosidase gene in frame, however, this surely could only happen in 33% of cases. Any thoughts?
Cheers,
Scott
The degradation of the T-overhangs leads to a blunt self-ligation of the vector, with subsequent shift in the reading frame of the beta-galactosidase gene and that's why resulting colonies are white. What I really don't understand -maybe it's out of the present topic- it's how is it possible that blue colonies arise if insert has not been ligated in the vector. In this case, the vector should stay linearized, isn't it? And also if transformed, it should be degraded immediately by cells, isn't it? And thus cells should not be able to grow on antibiotic medium! Thus: what are the blue colonies that we see on plates???
Thank you!
Cheers
W.
Hi Wally and others,
I don't have any definitive answers, however, I can probably suggest a couple of things to explain blue colonies.
1/ Starting material may not be 100% what it is supposed to be. I think this is fairly likely as we know most techniques aren't 100% effecient. Also if you place your T/A cloning vector without ligase you will see a few blue colonies.
2/ You can force ligations even with unfavourable ends to ligate and therefore even if you don't have compatible ends you can still get product. When the plasmid is copied these nicks etc. are corrected. This may also explain that when you add ligase to your starting vector you do see an increase in the number of blue colonies.
Just a couple of ideas, but no definitive answers. This question has troubled me before.
Cheers,
Scott
Dear all,
Well it´s been some time now since I posted my problem...it sure had some response!!! Thanks to all who took the time to read...
Now, I´ve had some advance with my problem: I checked 60+ white colonies with Colony-PCR and FAST (a quick lysis method) and only got 3 clones (3!!!) positive for the insert.
I think that the possible causes have been discussed here thoroughly...but one thing is certain: I´ve got to optimize my ligations...
Well, only 3 genes left to clone...see yall later....