DNA Isolation from Mouse Tissue - (Sep/17/2012 )
I really need assistance with extracting DNA from mouse tissues (tail, liver, spleen, kidney, thymus, etc.). When I remove the tissue, I crush it up in media (RPMI+10%FBS), filter, and then spin it down. I then resuspend the cells in 500 ul of a solution that contains proteinase K, and proceed to incubate the cells+solution at 55 deg overnight (shaking). The next day, I proceed to do a normal phenol chloroform extraction, sometimes twice to remove all the debris, and dissolve the final material in TE pH 8.0 and I added some RNase A. My issue is that sometimes I receive no pelleted DNA, or my yields are very low. Does anyone have any suggestions or a different technique that might yield more consistent results?
The crushing step is probably unnecessary, if you have bits that are no bigger than 5 mm on each dimension, then they should digest fine in the prot K overnight. I would just use PBS or the lysis buffer if I was going to crush them.
You should spin the debris down before the phenol-chloroform step(s), and make sure that the salt content of the buffers is enough to precipitate the DNA efficiently when ethanol or IPA is added. Add the RNase A before the P:C steps too.