DNA pellet won't go into solution - (Sep/10/2007 )
I am isolating Bacmid for transfection into insect cells. My pellets won't go into solution (TE).
What should I do?=?
ok bacmid are tough. First gently wash pellets with 70% ethanol at not too igh speed to ensure good salt wash and partial hydratation. I mean 80% ethanol does not help to the resuspension step. Seem you've overdried your pellet as it encounters difficulties to resuspend. But : do you have a gel-like pellet ? it may be RNA.
So try at first heating 55° 10' and see if it dissolves. If you still see pellet, go for 65° 10', check OD and run an aliquot on agarose gel to see if you don't have RNA. In taht case redo RNase, poteinase K and phenol chlo.
Thanks, but RNase should should take care of that right?The pellet doesn't have gel look, it is still like whitish flake. I read the OD and 260/280 was 1.91 and had 1.4 ug/ul DNA, which rules out protein contamination. I will run it on the gel. I tried heating @ 50 degree but wouldn't dissolve.
Normally, I incubate DNA pellet (Maxi, Midi) @ R.T until the pellet becomes transperant, but this time I did more than 10 mins (suggested by protocol) and did not see change in the appearance. So, I added TE and now the pellet wouldn't dissolve.
Leave it in the fridge overnight. Large DNA fragments can take hours to dissolve. If it's in TE then you could leave it at RT or 37C, but I wouldn't do that without some EDTA present.
Try adding warm TE 60C and leave it at 37C waterbath .