Dissolving plasmid DNA after precipitation

Rick Bright rbright at mnw.net
Thu Jan 23 22:19:02 EST 1997


Kevin,

I have this EXACT same problem and have tried the same things you have,
even Refrig overnight (which seems to help slightly).  Could someone
please offer some assistance here.  I know it is DNA in the pellet.  Am
I pelleting it too hard (12000 rpm @ 5') or am I letting it air dry too
long (15' at rt)?  Please Advise.

Rick Bright
rbright at mnw.net


>Kevin wrote: 
> Hi,
> 
> I often have problems dissolving plasmid DNA (either uncut or
> restriction fragments) following precipitation with sodium acetate and
> ethanol. The plasmisd and/or restriction fragments are anything
> between 1.5 and 10 kbp. Could anyone please give me any tips on how to
> succeed!
> 
> I normally do the following:
> - precipitate with 1/10 vol of 3M Na-acetate (pH 5) and 2 vols of 100%
> ethanol and leave for 1 hour either on ice or at room temp depending
> on the amount of DNA (the DNA is just as difficult to redissolve when
> precipitated at r.t. as when precipitated on ice).
> - spin for 15 minutes at 13,000 rpm on a microcentrifuge and the
> supernatant removed.
> - the tube is then filled with 70% ethanol and gently inverted a
> number of times to wash the salt out of the DNA, and then spun for 5
> minutes at 7000 rpm.
> - the supernatant is again removed and the tube re-spun for a few
> seconds to collect all the remaining drops at the bottom of the tube
> which are then removed (therefore no solution left behind apart from
> that left in the pellet itself)
> - the tube is then left with the lid open for anyting between 30 secs
> and 5 mins and then an appropriate amount of TE is added to dissolve
> the DNA (either successfully or unsuccessfully!). Leaving it to dry
> for 5 minutes was far too long and I seldom get any samples to
> dissolve. With the 30 secs air-dry I get more success but still it
> doesn't work all the time and I have to throw my sample away.
> 
> I have tried heating at 65-75°C for hours (even overnight) to get
> samples to dissolve but with no success. I have also tried doing 2
> washes with 70% ethanol to further eliminate any remaining salt, but
> again with no increase in the success rate.
> 
> Any advice would be greatly appreciated!
> 
> Thanks,
> 
> Kevin Mulcahy.



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