Colonies without inserts using pGEMT

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Claudia88
Claudia88's picture
Colonies without inserts using pGEMT

Hello, my name is Claudia and I am from Malaysia

I am having trouble with my ligation (or so I think). Once I have ligated at 16 degrees and left it overnight, colonies are formed on the plate (LB + ampicillin). However after running colony PCR, my insert is not to be found. I am using the pGEMT system. I am using a 1:8 ratio. 0.5ul pGEMT, 0.5ul ligase, 5ul 2x buffer and 4ul insert. I transform 10ul of ligation product into 100ul of TOP 10 E.coli competent cells. The cells are fine because I tried it out with the pRSET vector and the next day the LB ampicillin plate was filled with colonies

Could it be a ligase problem? I understand that the pGEMT vector is linearized and so if colonies are present, those cannot be the unlinearized vectors, right? I am using M13 primers when doing the colony PCR and as such after I have viewed the gel there are only bands present at ~250bp.

I am in dire need of help and any form of suggestions is highly appreciated

Thank you

Claudia

Nikail Collins
Nikail Collins's picture
cloning

It sounds like a PCR problem. Do all of your controls work for the PCR?  Can you tell me more about the process once you pick the colonies to run PCR on?