Dissolve my pellet, please!
gruther at bilbo.bio.purdue.edu
Wed Feb 5 02:00:33 EST 1997
In article <32F7A202.212F at codon.nih.gov>, jberke at CODON.NIH.GOV (Josh
> 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?
> Josh Berke
Hmmm....This surprises me a bit, since I've produced cDNA by RT that went
in just fine. Nonetheless, what I would do is increase the volume to 0.5
ml TE. If that won't get it in, nothing will; add a microliter of
glycogen, and precip with 3m NaOAc(pH7.0) and EtOH, wash and dry. Then try
to resuspend in a convenient volume. You may have some weird contaminant
that's causing probs that the reprecip will remove. If not, I'm clueless.
Alternatively, you might consider increasing the volume to resuspend and
dialyzing against your buffer of choice and then doing the reprecip.
Nothing worse than an annoying precious sample:) Good luck.
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