Hello, I am working on implementing DNA extraction from animal tissue using magnetic beads. The concentration of the extraction ranged between 1000-1500 ng/µl, and the A260/280 ratio was 1.85/2.0. However, when I run the gel electrophoresis on agarose gel, very intense bands appear, possibly indicating RNA, and no high molecular weight bands suggesting the presence of DNA are visible. The PCR works normally with this extracted DNA. I would like to know if anyone has had a similar problem. Could the RNA issue be resolved by treating the sample with RNase A (20 mg/ml) before adding the beads? If so, can the beads purify the DNA from the added RNase A? Additionally, what could be the reason for not seeing a high molecular weight band, indicating successful DNA extraction?
Hi Chrystian!
Can you tell me the size of your target DNA? All the other wells show very fine bands. Is this issue related to certain samples only?
Hi Sara!
On the top of the gel there are the PCR products from the extracted DNA, which worked fine. On the other hand, the extracted DNA are the first and second well, at the bottom. They do not show a band, only a smear. When I treat it with RNAse, the smear reduce but when I do not perform the RNAse treatment the smear significantly increase, suggesting that most of it is fragmented RNA.
The problem is, although the PCR seems to work fine, none of the extracted DNA shows a band on the gel, only smears
One of the reasons for having smears is an unoptimized PCR. Try to use different annealing temperatures i.e. a gradient PCR to get better results. Also run the extracted DNA (before PCR) alongwith the PCR product on the gel for comparison. This will help you to identify whether the problem is related to the extraction protocol or the PCR.
Hi Sara. The problem insn't with the PCR but with the extracted/genomic DNA that shows no bands in the electrophoresis (first and second well at the bottom)
It would be simpler to incubate the reaction mixture with alkali, then extract the DNA - no RNA should be visible upon electrophoresis.
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