QUESTION about cloning - (Jun/02/2010 )

hi all
i used fermentas TA cloning vector(pTZ57RT) for my job.
after transformation i see three type colonies
blue, white and white colony with blue center.


can you please explain about this?


regard...
iraj

White colonies mean that the LacZ gene has been disrupted. This usually means that some DNA fragment has been ligated into your vector. One hopes that this DNA fragment is your PCR product (since this is TA cloning). But it could be some random bit of junk. You have to check the plasmid.

Blue colonies mean that the lacZ gene in intact. This means that the colony has an empty vector, which is of no use to you.

White colony with blue center usually mean a poorly functional lacZ gene. This often indicates an empty vector that has been badly religated. Again such a vector is no use to you. However it could also indicate that vector has picked up a small insert (which has landed in frame with the lacZ gene.)

In the very rare situation where your insert is tinny, and by chance has landed in frame with the lacZ gene, such colonies will be your positives. But this is very very rare. You can check on computer if your insert is in frame with the lacZ.

Faced with blue, white and white with a blue center, pick only the white colonies for screening

thank you...

sometimes incubating the plate in 4 centigrade degree for an overnight can help to discriminate between white and blue colonies, this way pale blue and half blue colonies may turn complete and distinguish blue so you can pick the whites more confidently.

mahsa on Jun 20 2010, 05:32 AM said:

mahsa on Jun 20 2010, 08:32 PM said:

molstudent on Jul 5 2010, 02:25 AM said: