Hello,
When I clone multiple fragments into a vector, I get a positive colony PCR. However, when I then purify the plasmid and try to amplify the fragments, they are not coming up in the correct size. Has anyone had this issue before. I have repeated this multiple times, and after each positive colony PCR the desired fragments almost disappear?
When I also try to clone a gene (MEP1B) into a pET30 vector backbone, I also get a positive colony PCR and then when I try to re-amplify it does not come up (I have been successful with other genes into the same backbone)
Any suggestions or alternatives I could try?
Thank you!
You need to provide more information. What is the correct size of your fragment?
How much matrix did you use for PCR? Is it the same for colony PCR?
Sometime when using too much matrix the gel shows too weak bands.
Vanessa Sewell The colony PCR image which you have uploaded contains many non-specific bands along with your target amplicon. You can optimize colony PCR by changing annealing temperature, optimizing primer concentrations. Try to include positive control for PCR reaction. Positive control might be purified genomic or plasmid DNA. In another way, confirm presence of amplicon ligated in plasmid backbone by restriction digestion using two enzymes.
Regards,
Ajay Pathak
It is possible that your cloning failed and that when you plated out the cells there is wetting of the cells and agar surface with cloning reaction mix and that this was amplifying in the first pcr but when you grow on the colonies there is no insert so no amplification. This might be the case if your primers are internal to the insert but a pcr with one primer in the vector and one in the insert is a better combination to detect insertion
Dear Vanessa Sewell
How was going on with your experiment? I have the same problem in my cloning process. I would be grateful if you could share your experience to see what was the reason and how you solved it.
Hello everyone, I have not had any luck with this issue - this has happened with two genes now when cloning into a backbone. I usually do a colony PCR, then I re-PCR the positive colony with the exact same conditions and the gene seems to disappear. Could the gene of interest be toxic in some way?
What do you Re-PCR? is it the product of the first one, or the positive colony? and was the matrix " amount" the same in both reactions?
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