I bought a Lonza nucleofector transfection kit a few months ago, which has a pMaxGFP plasmid in it. Then we want to use this plasmid as the reference gene expression in some other experiments and therefore, the plasmid was transformed into the E. coli strain DH5alpha and preserved for further utilities. However, when we did the electroporation recently, we used the newly extracted pMaxGFP from our E. coli, and interestingly, there were cells (Raw264.7) expressing GFP 24-hour post-transfection, and the bacterium was completely okay in the kanamycin-contained LB (pMaxGFP has a kanamycin-resistance gene), even the concentration was acceptable in the nanodrop. When we were trying to illustrate the problem 'how the ratio of coil-coil structure in the plasmid may affect the transfection efficiency', there was completely no band on the 1% agarose electrophoresis. We first considered whether it was DNase contamination within the plasmid extraction kit, so another bland new kit was used and the whole experiment was repeated. Unfortunately, there was still no band. I just wondering has anyone done something similar and suffering the same issue as we did?
the attached image is the gel I'm referring to. from left to right: ladder (10k, 8k, 6k, 5k, 4k, 3.5k, 3k, 2.5k, 2k, 1.5k, 1.2k, 1k, 0.9k, then each band downward -0.1k till 0.1k), water (as -ve), the plasmid from Lonza kit, our extraction plasmid by new kit, our plasmid from the old kit, ladder again. all dosage as ~100ng/sample.
Many thanks!
Andy
I would subcline your transacted dh5alpha on kan plated and confirm presence of plasmid using colony per, then select a good clone to expand. I would also recommend using the kan at 30 ug / ml or at least 2x what you are using now to make sure it has not gone off.
There could be a number of different technical problems that could produce this result. First you need to confirm your plasmid is what you said it is, check if your bacteria which you transform is OK, try different ratios of plasmid and transfection reagent ... . Next you need to confirm you are extracting your plasmid with the correct kit and use a recommended protocol, this could be just a bad extraction problem. I would recommend heat shock as the method of transformation which I found to be the most effective one.
Your gel is not very well stained, or your picture is underexposed significantly. I think you are missing lots of possible things in the gel for that reason. But assuming you still don't see DNA, try to repeat the preps before worrying too much. Sometimes it's just a trivial technical issue. If you are seeing GFP signal you should have plasmid.
Lora Benoit Mateo Glumac Michael J. Benedik Thanks for your recommendations. I tried another plasmid (pmcherry-N1) today, which is ~4.7kbp in size. When the plasmid was uncleaved, the position of the band was over 10kbp. But after EcoRI cleavage, the size of plasmid is between 4-6kbp. So I suspect whether running the plasmid on agarose gel is an appropriated method to examine the plasmid quality?
Hi Andy, typically, uncut plasmid will run faster on an agarose gel compared to linear plasmid, appearing lower molecular weigh.
To confirm that the plasmid is the correct weight, it must be digested with a restriction enzyme that cuts once and run out. So it appears this is working for you. However, I am not sure why your uncut plasmid is running at a higher apparent molecular weight.
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