I am trying to isolate a 13kb DNA fragment from an agarose gel. I use freeze thawing, phenol/chloroform extraction and I tried precipitation using either 100% ethanol or isopropanol. In all instances I see a pellet after precipitation, but I measure no DNA after washing the pellet with 70% ethanol and dissolving it in water. I am 99% certain the pellet was not lost during these steps, but a nanodrop indicates no DNA. Is it possible that the pellet is something else, or that I did lose the DNA during one of the steps?
Wow: what % agarose did you use?
I would check E260 before P/C extraction to be sure there is something there to extract!
Nanodrop may be faulty: test with known DNA?
If the volume after P/C is low, there is a lot of salts in the aqueous phase and you add more than 2 vols of ethanol, the salts may have precipitated, and be washed away in the 70% ethanol.
Co-precipitation with glycogen (usually 1mg is enough) will improve yield.
Try a few things:
1) add 70% ethanol to wash, agitate and then spin again for min. 5 minutes.
2) add 1ul of glycogen
3) check the pH after water adition
4) measure concentration with a fluorometer
Check your steps and do them properly. Final step is to add 100% Etoh is to make final concentration of Etoh around 60% in tube (bc in the tube there must be liquid after removing of cell debris). Then you have to centrifuge the tube 11,000 g x for 5 mins, best in 4 degree C centrifuge. Take the tube gently, get rid of supernantant. Then you can do 2nd wash with 60 to 70% alcohol or not washing also OK but have to do drying properly to let all alcohol is gone away. Either use vaccum centrifuge 4 degree 10 mins or just open the cap of tube and leave it in room temp until all Etoh vapourize.
then only dissolve with DNase free with RNase+ H2O and load it into well of gel. Check.
I think your DNA was gone with Etoh when you wash it.
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