Dissolve my pellet, please!
jberke at CODON.NIH.GOV
Tue Feb 4 15:51:45 EST 1997
I have pellets of cDNA that just do NOT want to dissolve.
I performed a phenol:chloroform extraction followed by
a phenol:chloro:IAA extraction. I precipitated the DNA using
4M NH40Ac and 95% ethanol, with 20min room temp spin at 14,000.
This was done in a siliconized 0.5ml tube (from Ambion). After
removing the supernatant I could see the pellet; the cDNA is also
radiolabelled so I can keep track of it.
I then added 500ul of 80% ethanol and left it overnight at -20C (I
thought this would be a suitable pause point in the protocol). The
next day I spun for 15 min at 14000 and removed the supernatant.
I let the tube air dry for 10-15 min; the residual ethanol
evaporated but the pellet still appeared moist.
I added 10ul of water and pipetted up and down. Did not dissolve.
I heated at 65C for 5 min. Did not dissolve.
Removed water, added fresh 20ul water. Vortexed heavily.
Heated two hours at 65C in a thermomixer (continous agitation).
*Still* did not dissolve.
Left at -20 overnight
Next day, removed the water again, added 20ul TE, left at
room temp for 8 hours. Still did not dissolve. The tube remains
hot but the added liquid does not.
Unfortunately this is quite a precious sample. Any ideas?
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