I am conducting an experiment using the results from pMD2.G and psPAX midiprep Q prepared by a senior in 2014. The process involves transformation, midiprep, and midiprep. However, despite adjusting the DNA concentration to 20-30 ng/μL and using 1 μL mixed with 10 μL of DH5α before plating, no colonies are forming. Since colonies form well with the positive control (pUC19), it doesn’t seem to be a plate issue. Could the problem be due to the DNA being too old? Should I increase the DNA concentration or change the amount of DH5α used? Here is my experimental procedure:
If stored correctly, old plasmid should be good for years or decades. First, I would run some on a gel and be sure you can see plasmid DNA. If the DNA is degraded you would still get an A260 even with no intact plasmid.
Secondly, when you say you got colonies with your positive pUC19 control, did you get a LOT of colonies? You should have thousands or even hundreds of thousands. If not, then maybe your competent cells are not really that good any longer.
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