I am trying to transform DH5-alpha E. coli with plasmids containing a desired gene along with the amp resistance gene. I have done this exact procedure several times before with the same plasmid (containing different desired genes) with no issue, but this time, I am getting no colonies at all. My competent cells seem to be fine as the cell aliquot transformed with my positive control plasmid grew fine.
My protocol involves incubating 20 uL aliquots of competent cells with 10 ng of DNA for 30 minutes on ice, then heat shock at 42.5 degrees C for 40 seconds followed by incubating on ice for 2 minutes. Then, I add 250 uL of pre-warmed LB broth without antibiotics and incubate in a shaker incubator at 37 degrees C for 1 hour. Then, I plate 100 uL of the mixture on plates containing ampicillin and let the plates incubate overnight at 37 degrees C.
Any suggestions would be appreciated.
Jeevan,
You could try one of these:
1. make sure the drug concentration is correct
2. Use the whole volume of transformed cells for drug selection
Keep me posted.
If this is pure plasmid (and not a ligation mixture) then there really are only a few possibilities. 1) competent are no longer good, but it seems as if you did a positive control so probably not this; 2) plates are incorrect (are you certain about the drug resistance of this plasmid?); 3) some error during the protocol (again unlikely if you did positive control at the same time); 4) no plasmid DNA (take a look at your plasmid on a gel)
Hello,
in addition to what has been proposed to you as solutions, I suggest, if the problem is not resolved, to start with a preparation of competent bacteria intended to be transformed by electroporation.
Electroporation is more efficient than heat shock transformation of DNA, however, you need to be sure that your DNA does not contain salt
My best guess would be a DNA issue, as your cells give you colonies from your + control (that rules out cell issues, plate issues, incubator issues). Try re-quantifying your DNA and, based on that number, run a visualizable amount (50-100 ng minimum) on a gel to make sure it's not degraded.
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