Hello everyone,
I use several pcDNA3.1 expression vectors to transfect cells.
The vectors were prepared by midi-prep a year ago and diluted in TE buffer.
Now that I run new experiments, I decided to measure plasmid concentrations again, prior to transfection.
All their concentration have droped by 2 to 3-fold.
260/280 ratio are still good (over 1.8), but strangely 260/230 ratio have risen (from 2 to 2.3~2.5).
Given the good 260/280 ratio, the presence of EDTA in the buffer and the -20°C storage, I'm pretty sure it is not degradation.
It could be adsorption of DNA on eppendorf tube wall but given the 100~500ng/µL range of concentration, I don't think any tube surface could sequester this much vector quantity.
Anyway I heated my vector for 15min to 60°C and votexed it without increasing the measured concentration ?
The only thing I see would be freeze/thaw cycle maybe ? (I did 10 to 20 such cycles...)
Should I add glycerol to my TE so that freezing and ice crystals don't shear my vector ?
Or just aliquot my vector?
Where did my vectors go guys ???? ^^
Thanks for the help you can provide,
Philippe.
I think you should double check your spectrophotometer, did you use the same one? Was it blanked correctly both times?
I doubt that much happened to your plasmid, even with freeze thaw cycles the worst that would happen is the DNA is nicked or cut, but that would give you the same 260 reading. Even if converted entirely to nucleotides I think the reading would stay essentially the same (or even go up slightly). So that is why I think it may be an issue with your measurements.
Thanks Michael J. Benedik ,
I had not even considered that the apparatus could be the issue.
I'll try measuring the concentration with qubit to be sure.
But remember it might be the original measurement that was off. Lastly it might not matter, use the lower concentration as real. and It won’t hurt if there is a bit more than expected Unless you need to be very quantitative
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