I used the Dneasy PowerSoil Pro Kit. But I didn't get a good ratio of 260/280 and 260/230 and didn't get a good band after electrophoresis. I maintain all procedures according to the manufacturer's protocol. What should I do now? Need your valuable suggestions.
A low 260/230 ratio may indicate the presence of contaminants that absorb at 230 nm or less, such as EDTA, carbohydrates, phenol, or guanidine HCL. A low 260/280 ratio may indicate the presence of contaminants that absorb at 280 nm or less, such as proteins or phenol.
I would try adding 1-2 extra desalting wash steps (I think it's buffer C5 in that kit?) and leave the buffer sitting on the column for ~3-5 minutes each time and see if that helps. In my experience following the manufacturer protocols for nucleic acid spin columns to the letter at the desalting steps results in bad ratios (particularly 260/230) on many samples. It takes time for the residual chaotropic salts added at the first stages to dissolve fully off the silica membrane into an ethanol solution and they're not always all taken off with a single wash.
I'd also recommend eluting in a buffer with a low (0.1 mM) EDTA concentration in case there's stable nucleases in your sample. While a lot of places are paranoid about EDTA interference with downstream processing, I've never seen a concentration this low cause any real issues.
If neither of those things help then hopefully someone who's more specifically worked with the same types of samples as you can help.
The other possibility to consider is that you just did not recover very much DNA. If you have little DNA then it is easy for low level chemical contaminants to swamp your 260 ratios.
You may follow these additional steps after extracting the DNA using the DNeasy PowerSoil Pro Kit. This will enhance the purity of the extracted DNA. I believe you will find a decent 260/280 and 260/230 ratios. The attached photo shows the comparative findings of my research.
Step 1: Add 0.1 vols of 3M Sodium acetate (pH 5.2) to your eluted DNA, and 2 vols of ice-cold 100% Ethanol (molecular grade). Mix the solution by pipetting slowly using aerosol-resistant pipette tips.
Step 2: Keep this solution at –20°C for overnight.
Step 3: Next day, centrifuge the sample at 14000 rpm for 30 minutes at 4 degrees Celsius. [Prior to centrifugation, remember to mark the side of the tube where the pellet is expected to be and don’t let it out of your sight when decanting the ethanol. You may hardly see the DNA pellet in the tube, but DNA is still present in the tube as a pellet].
Step 4: After centrifugation, carefully discard the supernatant without disturbing the pellet.
Step 5: Wash the pellet by adding 200 microliter of 70% ethanol. Gently invert the tube 2/3 times.
Step 6: Centrifuge the tube (with 70% ethanol) at 14000 rpm for 30 minutes at 4 degrees Celsius to pellet the DNA.
Step 7: After centrifugation, carefully discard the supernatant without disturbing the pellet.
Step 8: Keep the lid of the tube open for 10 mins inside the laminar airflow cabinet to completely air dry the residual ethanol.
Step 9: Add 50-100 microlitres of elution buffer (C6 solution)/TE buffer/Nuclease-free water in the tube and gently solubilize the DNA pellet by pipetting.
Step 10: Measure the DNA quality by NanoDrop and store the DNA at a -20 degree freezer for further use.
Thank you!
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