I performed a standard PCIA extraction (2x PCIA, 1X chloroform) on my PCR samples to clean them up for use as an enzyme substrate. By A260, (nanodrop) I later found I have overestimated the [DNA] ~6-fold as judged by staining on a gel with another sample as standard (speced on the same nanodrop). What can explain this? I thought maybe dNTP carryover but this seems excessive. I also tried Qbit assay and it was very strange, with 5x diluted samples giving the same very low reading as undiluted samples (control sample estimated concentration fine). The PCR fragments are cut by REs fine.
Ant ideas?
thanks
Dont think its dNTP carry over, especially after wash steps and silly question but the PCR solution, properly mixed after being frozen? Sure it was but always good to ask.
I would personally take the nanodrop as more accurate than gel. I am assuming the standard with a known volume spec'd correctly? and when the gel was run there was no fragmentation??
Hello
How is the shape of the nanodrop curve? Also how was the purity ratio?
Hi thanks for responding. The extracted DNA 260/280 was normal (around 2). On the gel I was compared a column cleaned up fragment and also a plasmid. Both were speced on the same nanodrop and both stained ~6x more than what should have been an equal mass of several extracted fragments. So its something about the extraction but dont know what
There could be something which have the same absorption as DNA at 260, could be residual phenol or could be something with binding capacity with DNA.
Please look at this post: https://www.researchgate.net/post/High_absorbance_of_protein_at_260_nm_but_not_because_of_DNA_Why
For low amounts of DNA better to use fluorescence based quantification, including agarose gel+fluorescence staining, http://seqanswers.com/forums/showthread.php?t=21280
Hi William,
At least in our hands, Nanodrop is notorious for overestimating the DNA quantity, unless the samples are very pure, such as those that have been passed through columns. The quality readings (A260/A280, A260/A230, etc.) from the Nanodrop are OK.
It is better to use gel estimation, running your sample side by side with a ladder with known band masses. Dye based quantification (e.g. Qubit) should be used if you want more accurate estimation. If your sample concentration is low, use the High Sensitivity option of the Qubit.
Hope it helps and good luck!
Best,
Sheng
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