Hello! I used 5uL of my samples to do a colony PCR. It showed no bands on the agarose gel, but showed bands that corresponded to the purified plasmid and a blue colony . First, i just though my insert wasn't there, but after isolated and purified the plasmid and then digested with restriction enzymes for 3 hours, I get bands of these colonies which are with the expected sizes of my insert DNA. What could be the problem?
Thanks!
It sounds like either you are using old/the wrong primers or that your pcr annealing temperature is too high so the pcr is failing
Hi Isabela,
I am sure that the problem is with your pcr reaction. You have used 5microliter of culture which is too high amount of template. For colony pcr actually one or two cells are enough, as single cell contains multiple copies of plasmid with insert and thus pcr reaction get inhibited with high template concentration. You should use less than 0.1 microliter of culture in 10 microliter of pcr reaction, I am sure you will see amplification and band of your expected size.
Similarly you can do pcr with your isolated and purified plasmids to check presence of insert. For such pcr with plasmids as template use 10-20 ng plasmid as template. Just cross check the doubt regarding primers and annealing too as mentioned by Dr. Paul. I am sure you will be successful and can save time reqired in restriction digestion in your future experiments.
Colony PCR is a crude reaction and doesn't always work. Sometimes you have to do the longer protocol to get good results. In this case, grow an overnight liquid culture, do a plasmid DNA prep, linearize with a single-cutting enzyme, THEN do PCR. You can't rush quality. Good luck!
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