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Gentle method to dry a DNA pellet after alcohol precipitation


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5 replies to this topic

#1 Ikar

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Posted 29 May 2011 - 01:50 AM

Hi,

I am looking for a quick but gentle method to dry a DNA pellet after treatment with alcohol (e.g. after Midi preparation or DNA extraction from other tissues).

My approach was to pellet the DNA and then I decanted the supernatant alcohols and put the reaction tube on a liquid soaking paper - upside down.
After a couple of minutes (30 or so) I lay them down horizontally so the alcohol cto allow the alcohol to evaporate.

But this method takes about and hour or so, anyone knows a faster method (except for putting the reaction tube on a 50+C heating block)?

#2 hobglobin

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Posted 29 May 2011 - 01:56 AM

A speedvac, if it's gentle enough for you (vacuum, moderate heat, moderate centrifugation speed).

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#3 Trof

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Posted 29 May 2011 - 11:47 AM

Instead of decanting the ethanol wash, we centrifuge the last step with the opening of the tube pointing down (so we know that the pelet would be always on the opposite side of the tube) and then pipette all the ethanol out slowly and watch for the pelet if it's visible. If the pelet isn't visible just act as if it's on the wall were it should be. You can pippete everything out, because pelet is on the wall and not on the bottom. We use a new tip if there is some small residue after filling the first one. And of yourse don't leave drops on the wall. After this we let it air dry in the flowbox for 10 minutes maximum, till the pelet's clear.

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#4 Ikar

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Posted 29 May 2011 - 01:12 PM

Instead of decanting the ethanol wash, we centrifuge the last step with the opening of the tube pointing down (so we know that the pelet would be always on the opposite side of the tube) and then pipette all the ethanol out slowly and watch for the pelet if it's visible. If the pelet isn't visible just act as if it's on the wall were it should be. You can pippete everything out, because pelet is on the wall and not on the bottom. We use a new tip if there is some small residue after filling the first one. And of yourse don't leave drops on the wall. After this we let it air dry in the flowbox for 10 minutes maximum, till the pelet's clear.


This sounds very interesting. But either I didn't follow that or you have very special centrifuges that allow you to put a 1.5 reaction tube with the opening down.

#5 Podge

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Posted 07 June 2011 - 07:44 AM

When extracting RNA we do it as follows:

Wash with 75% Ethanol (1ml) and then after the centrifugation we suck of all the ethanol. P1000 for the majority of ethanol, then a P200 and then the last bits with a P10. If there is drops on the side I either collect them together and suck them off or if they are to little to pipette I smear them on the wall so they become very very small and evaporate easier.

Then I let it sit open and on the side for about 10min. The pellet becomes more white then. So you see that it starts to dry.
Then resuspend, let it sit for 2-3min and then mix.

Do you use GlycoBlue? Makes it waaayy easier as the pellet becomes nicely blue.

#6 phage434

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Posted 07 June 2011 - 11:30 AM

What Trof was trying to say is to rotate the tube (still upright) so that the pellet is towards the center of the rotor, rather than away from the center. Then gently centrifuge to put all of the liquid away from the pellet. I do this with a small 100 cm low speed bench centrifuge, rather than a large high speed one. He was not suggesting putting the tubes in upside down.

You need to be somewhat quick about removing the liquid, since as the ethanol evaporates, you are left with water, which will dissolve your pellet. You want to remove the liquid before this happens. In general if you remove all the liquid with a 10 ul pipet tip after centrifuging, you can just leave the tube open on the bench for less than 10 minutes to evaporate the ethanol. You can tell when this is done by smelling it.




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