Hello. We are trying to optimise an "heteroduplexing" protocol based on another paper. TL;DR we want to denature a mixture of DNA through drastically increasing pH, then neutralise the pH with a buffer (expecting the DNA strands re-nature randomly) and then perform DNA ethanol precipitation. In the end, we end up with a very high yield of DNA but with an extremely low purity: a 260/230 ratio of 0.5. But what worries me the most is the absorption spectrum (see attached image). It has no peak at 260nm at all, not even a "shoulder"... purity of DNA is not important for our downstream experiments but looking at this spectrum, I wonder if we have DNA at all. How can I verify if we have DNA in there?
The protocol we did goes as follows:
1. Add 500 mM NaOH (pH 13) and 1.5 M NaCl (increases pH)
2. Incubate 5min
3. Neutralize by adding 25-fold volume of 100mM Tris-HCl (pH 8) and 10mM EDTA
4. Incubate at 68 degrees for 3h
Precipitation phase:
Thank you in advance.
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