When I isolate DNA from cervical cells in EasyPrep liquid (cervical swabs), the DNA amount is very little in spite of a large quantity of cells (a big pellet at the bottom of a 1.5ml tube). The amount of DNA I got is less than 10 ng/uL, sometime is less than 1 ng/uL. I also try washing cells with 1X PBS (5 times) but the results are still the same. Therefore, the PCR product of GAPDH gene has no band or a pretty blurry band. The kits I use here include E.Z.N.A.® Tissue DNA Kit, E.Z.N.A.® Blood DNA Maxi Kit. However, when I use Kaneka easy DNA extraction kit or even I just wash cells and add cells directly to a PCR reaction, the PCR product of GAPDH is really clear.
Can anyone help me explain this situation? Whether maybe any composition of Easyprep liquid (its components are not disclosed) affect the ability of DNA to bind to the silica membrane of the column?
Thanks so much.
First check that the zeolite resin in the columns is not cracked. Consider leaving your lysate in the column for 5 mins to allow the column membrane to equilibrate. Put your lysate through the column twice. After the ethanol containing wash step empty the waste catchment tube and centrifuge again. Leave the columns for 10 mins to allow for ethanol evaporation before eluting. Elute with warm water/buffer. Leave the eluant to soak into the membrane for 2 mins and put the eluant through the column 2-3 times.
Did you check if everything was stored properly? Maybe do the same isolation with the DNA easy prep liquid from a kit of your colleagues. Did you measure the DNA concentration with nanodrop? How were the curves?
you can use other method, but anyway you have to use higher concentration proteinase K. I think its a key for bad recovery/
Thanks Robert Adolf Brinzer for your advice. Also, the resin in the column is absolutely intact.
Hi Verena Gschiel , the kit was stored very well. I also used it for isolating DNA from other materials and the DNA yield was high, above 200 ng/ul. In addition, I measured the DNA conc. with nanodrop and the curves were also bad as the yield.
I will try using higher concentration of protein K. Thanks Ahmed Darweesh Jabbar Al-Rubaie
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