DNA concentration not stable over time - (Mar/20/2006 )
I measured significant variations in DNA concentration of my plasmid preps over time.
I typically prepare a lot of plasmids, after which I then transfect into cells.
Plasmid extraction kits used are the Qiagen ones (Maxiprep and Megaprep).
I normally measure DNA concentration right after resuspension in TE. I believe that this DNA is pretty clean, but I observe that after some time (less than 1 month !), the DNA concentration can be as much as 50% less than the first time. (Other people in the lab have the same problem.) DNA is stored at -20C and is thawed before use at room temperature.
The spectrophotometer we have is a small bench machine (Grape Ape, Ultrospec 2100 pro UV/visible).
Does anybody know anything about this ? Is it simple degradation or a spetrophotometer problem?
Any help would be really appreciated.
1. DNA is probably being absorbed to the tube walls. Siliconized microfuge tubes will reduce this effect.
2. We have aliquots of a prediluted primer frozen at -20C that we use in our monthly calibration check of the spectrophotometer. The average over the past 4 years is 115 uM, and we usually get a number between 105 and 120 uM, but the range is between 95 and 135 uM.
3. Fluorometer readings with Hoescht 33258 are more reliable than spectrophotometer readings.
Thank you for your answer.
Do you know by chance, if absorbption by tube walls has been quantified or estimated by someone ?
I am sure that our spectrophotometer is not very accurate and I am not surprised by weak variations, but 50% is really a lot.
My feeling is that something (polysaccharides...??) that absorbs UV precipitates after several cycles of freezing/thawing. After resuspension of my DNA, I observe sometimes that my samples are slightly cloudy, but this can be removed after centrifugation. This seems to be normal. Probably there is still something ...
In a test I performed 6 years ago, I kinased a 100 nucleotide RNA molecule and cleaned up the reaction on a G-50 cartridge. The concentration in microfuge tubes from numerous manufaturers and several lots was 0.4 nM (2.4 e 14 molecules) in buffer containing 137 mM sodium. After incubation at room temperature for 1 hour, the RNA was removed and counted in scintillation fluid. Non-siliconized tubes retained 25% of the input RNA. Siliconized or non-stick tubes retained 5%.
Countless numbers of ethanol precipitations of higher concentrations of RNA and DNA since then have confirmed this result. Ethanol precipitation with ammonium acetate or sodium acetate in siliconized tubes results in 5% retention. Non-siliconized tubes retain 25%. Falcon or Costar 15 and 50 mL tubes behave as if they are siliconized by a mold-release agent.