Hi everyone,
I have extracted some plasmid DNA (pCMV2-SEP-GluA1) using the ThermoFisher GeneJET Plasmid Miniprep kit. Nanodrop suggests that there might be some RNA contamination (A260/280 ratio is 1.97), but a good yield was obtained (3.8 ug/ul). I would like to use this DNA for transfection of primary neurons (from the embryonic rat). Is it necessary to purify the plasmid further for transfection or can the the product from the Miniprep kit be used directly to transfect my cells? I plan on using two different methods for transfection: the calcium phosphate method and Lipofectamine 2000.
The Miniprep kit is a quick and easy method for isolating plasmid DNA from bacterial cultures. It is relatively inexpensive and produces high-quality plasmid DNA suitable for most transfection applications.
However, Mini-prep may not be suitable for all transfection applications or produce as pure plasmid DNA as other methods. It is more time-consuming than other methods.
Yes, you should purify your plasmid DNA before transfection, as contaminants like proteins, salts and other nucleic acids can interfere with the transfection process.
yes, I would recommend purifying your plasmids. Ideally one should purify plasmids before transfection, more so in your case because you are going to transfect primary nurons (you would not want any molecule to dictate ambiguity to your results, primary neurons are derivative of very sensitive physiology). you would lose some yield in the process, but it's worth it.
if you are in doubt, and if it's possible, you can do two sets of experiments, one with purified plasmid and one with the plasmid you have. You probably should see virtually no significant difference, but there is always a chance for unexpected results. and if there is any difference between the two, the one with purified plasmid should be the result to move forward with.
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