Hi, so there's my problem. I have done a RNA extraction, ran a gel with it and dosed it. The picture of the gel is after my message and for all my samples, the concentration was between 1250-1550 ng/ul, ratio 260/280 between 2.09-2.13 and ratio 260/230 between 2.19-2.30. I thought this was excellent but my coworker suggested me to run a PCR to verify if I have DNA contamination. I ran a gel after and there's amplification in all the wells (the positive control is not shown but there's a band exactly at this level). Nobody from my lab could answer my interogation about what to think about those results...
Dear Marc-Antoine,
Generally DNA contamination will appear up in the gel, as a band in the well. There where I put an arrow in your picture.
Hello,
Thanks for your answer, I will do another DNAse treatement!
Hello Marc-Antoine,
To complement the information: It is always safer to asume that you have DNA contamination, even if you cannot see the high molecular weight band in the gel. PCR is a sensitive technique and you will get amplifications, even with a little contamination. Both DNA and RNA absorb at 260nm, so there is not way of differentiate them with the concentration measurements (unless you are using a specific fluorescent-dye-based method, such as Qubit). Good 260/280 and 260/230 ratios tell you that you don't have other types of contaminants, such as protein or phenol.
Also, your RNA concentration is quite high so you should check the DNase protocol carefully to make sure that you are using enough enzyme. DNases have limitations on the amount of DNA that they can handle, so be sure to use enough.
Thanks a lot for your answer, I did another DNAse treatment and now the PCR is running so I'll get the results in about an hour!
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