Sequencing DNA - (Dec/25/2006 )
I have a simple question. For DNA sequencin I am using classical method for preparation of the sequencing mix.
After my PCR reaction, I visualize the bands on the agarose, and after that i am supose to cut only the wanted bands, not those resulting from primers or primer dimers. So, by now I am Ok. To extract the fragments from agarose I use ethanol precipitation with sodium acetate pH 5,2. My question is how many ml of this mix should I use, starting from lets sey 0,33mg PCR fragment in agarose gel. Becouse somtimes I get good yeld, but sometimes nothing. I dont think that i am makink some mistake in the rest of the protocol
It is possible that you are exposing for a long time the DNA to UV light, if the pcr product is big enough (eg more than 200kb) try to purify by column instead of agarose purification. When you precipitate with ethanol you could loose some product. But if the product is to tiny try freeze n squeze (don't know if a write it correctlty) from Bio Rad that use agarose purification with a column is super simple and have good results.
and also the ethidium bromide in the agarose won't do the DNA any good.