I have vertebrate tissue samples stored in DMSO for about 10 years. I would like to do RAD analysis in those samples. Is there a way to extract DNA with good quality for the analysis?
Have you ran out your extracted DNA on a gel? You want to have high molecular weight genomic DNA for RAD lab work. Essentially you're using 1 or 2 enzymes to cut up the genome, so if your DNA is degraded then many of your cut sites may be broken apart, and thus the enzyme won't recognize them. I could also imagine that if you're DNA is highly degraded then you could potentially end up with many fragments being smaller than your desired size range. So, even though the adapter would stick to them, they would be thrown out during size selection. I would suggest running out some DNA extractions on a gel to see if there's any high m.w. DNA and use a fluoremeter to check DNA quantity. If there is some high m.w. DNA, then digest some of that sample with your enzyme(s) and see if the resulting fragments are in your desired size range. Perhaps you could find someone in your dept who would be willing to let you throw a sample or two on their lane so you could just see what you get!
Dear Brittany,
I got to the conclusion that my old DNA was not good at all for RADs so I went to the field and got new samples.
Here are the two types of gel that I've been getting. I usually consider type A to be a bad example and get more happy when I get type B since degradation is almost absent. Notwithstanding, I've been getting really low 260/230 ratios from Nanodrop, can you give me some insights on these results? I would appreciate your opinion and expertise!
Thank you!
Hi Telma,
Sorry that I didn't see your message until now. Those samples look pretty good to me. I've done RADseq on DNA extractions that looks really similar to those and gotten good results.
I wouldn't recommend using a nanodrop for quantifying DNA...try to find a lab that has a fluoremeter (e.g. Qubit). Those give much more accurate readings.
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