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[hotstuffdb22]

Hello,
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?

Thanks!!!

[bob1]

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.