I got bacterial colonies after transformation, but all were negative after colony PCR when compared with positive control.I don't know why. If you know the problems, please let me know.
Your colonies might be false positives or having empty vector. Use blue white screening method for selection of transformed colonies having plasmid with insert.
Colony PCR is a not very accurate verification method, you can extract the plasmid for sequencing and see if the sequence is the one you want.
The more likely reason is that some details have been mistaken during the transformation process.
It could be also a problem with DNA concentration, I usually try to dilute bacterial DNA since I've had issues with using it directly into the PCR mix. I usually "touch" each colony with a tip, place it in 10 ul of sterile water and then use 1 ul from there as template for the PCR! That's only and idea, but there could be many reasons why you're not getting results.
Was your positive control pure plasmid DNA or did you also do colony PCR on your positive control? If you used purified DNA then you might be having an issue with colony PCR, so you would need to troubleshoot that process first. If that is working fine, then you should troubleshoot your cloning protocol. What happens when you ligate vector only without insert? Maybe you just had uncut vector DNA or lots of self ligation. So stepwise determine where the problem was.
Thank you all for suggestions. I think the problem may be in ligation step. Now, I start from the digestion step.
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