I'm doing stool DNA extraction using QIAgen DNA Stool Mini Kit! Most of my result shows 260/280 ratio higher than 2 (between 2-2,5). Besides 260/280 ratio, DNA concentration is also low (around 30-60ng/ul). I would like to inquire how to modify the protocol to solve those two questions? I had increased the stool to 500mg and changed the lysis temperature to 95, but still had no effect.
Anybody has some good advice to me? Thank you for your help in advance!
I am not aware of this kit but you could use more sample and lysis buffer in proportion to increased sample. in that case you have to add sample twice to the column. and other downstream processes would be same
hope this will work to increase your DNA concentration. as far as the ratio it may be due to RNA contamination. as you are increasing temperature so it may include the lysis of the pathogens present in sample. due to more number of pathogen you may getting more RNA while extraction. and this is ultimately affecting A260/280 ratio.
all the best
Hi,
Seems like you do have a problem with the quality of DNA. I never used the kit you mentioned above, but i hope basics may remain similar for the extraction. So i can provide you couple of suggestions you might like. First of all concentrations you had similar like RNAs, so RNAse treatment could be one solution. Again you didn't mention the 260/230 ratio so that i can tell you about an extra step which is washing of DNA solution using 70% ethanol. Beside if you do have any "Elution Steps" like other kits you should make your stay little longer before going for the centrifugation. Which might give you Improved concentration of DNA.
Best of Luck,
Imtiaz
Imtiazur, thanks a lot for your good suggestions!
Today I have tried to use RNase to treat my samples, and the OD 260/280 is around 1.8, but it seemed that the DNA also had been degraded, so the concentration decreased to 5 ng/ul.
The OD 260/230 is around 1.3-1.5, also very low. May I consult you again? I don't understand this step "washing of DNA solution using 70% ethanol". As I have added 100% ethanol to the two wash buffers as the protocol said. So what you mean is to add this step after the second wash buffer?
As for the elution steps, I have added the incubation time from 1min to 5mins.
Thank you again for your kindly help!
Hello Again Liu Fang,
Thanks a lot for marking my answer as a good one. My speculation was right regarding RNA contamination therefore RNase treatment got you a good 260/280 ratio. For that reason you don't need to take that 70% ethanol step. I told for that to make your DNA more pure. You mentioned about degraded DNA which decreased your conc., is not right indeed. Because if you use NanoDrop to check the conc. it will count the whole nucleotide content of DNA, so there is no chance to say this. Only you can say about degraded DNA after doing Agarose Gel Electrophoresis. Only by taking conc. you can’t say that your DNA is degraded.
260/230 ratio is also important to check whether there is any contamination of Phenol, EDTA, carbohydrates (as they have absorbance near 230nm). It is recommended to be 2.0-2.2. Lower than this implies contamination.
Finally, seems like now you have to deal with the concentration of DNA to improve. For that i can say you some words you might be interested in. First try to improve the volume of sample, second check the manual thoroughly if there is any troubleshooting they had mentioned regarding your problem, third check the quality of 100% ethanol you have used and in lieu of absolute ethanol I might prefer using ice cold Isopropanol.
Best,
Imtiaz
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