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Ethanol Precipitation


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5 replies to this topic

#1 xyne

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Posted 11 October 2011 - 08:28 PM

Hi there,

I'm trying to use this ethanol precipitation method to precipitate down my RNA. I added 0.8M (final conc) of LiCl and 2.5 volume of absolute ethanol into my sample and chilled it in -20 degree celcius for half an hour. After that i centrifuge the sample for half an hour up to 13500rpm. But i get nothing after all these procedures. Can anyone help me with this? Thanks.

#2 bob1

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Posted 12 October 2011 - 02:14 PM

In what size rotor? RCF or G forces are the only measure that is useful when talking about centrifuges.

The pellet may be small or transparent, and (should be) attached to the wall of the tube. If it is not attached, as sometimes happens, you may be able to see it floating at the bottom of the tube.

How much RNA are you trying to precipitate? If it is from a small sample, you may not be able to precipitate it efficiently without a carrier.

#3 phage434

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Posted 13 October 2011 - 03:33 PM

If you are having trouble with DNA or RNA precipitations, then adding Novagen Pellet Paint makes it easy to see the pellet. There are both fluorescent and non-fluorescent versions. I'd recommend the non-fluorescent, since the fluorescence can be a real problem with some downstream use such as sequencing. You probably will not have to do this more than a few times to get confidence that the pellet is there, even if you can't see it.

#4 xyne

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Posted 13 October 2011 - 10:53 PM

Bob1,

The G forces that i'm using is 9.6G. The RNA that i try to spin down is around 10μg. Is it possible to prolong the centrifuge time to 1 hour or 2 hours?

#5 xyne

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Posted 13 October 2011 - 10:58 PM

phage434,

Thanks for your opinion. I try to get more information about the Novagen Pellet Paint.Thanks a lot

#6 allynspear

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Posted 14 October 2011 - 06:08 AM

Xyne,

I know some people disagree, but I have found that with smaller amounts of nucleic acid, and particularly RNA, a longer incubation at -20 degrees does give me better results. I typically would go 2 hours to overnight with RNA at 10 ug or less. And I would also say that a longer centrifuging time can also help, so 1-2 hours may work, but that is dependent on the volume you are in. Are you using a 1.5 mL microfuge tube or are you in a much larger volume?

Lastly, why are you using LiCl for your precipitation? I know that it is a common technique, but you need to be very careful about getting rid of all of the residual Lithium using 70% EtOH washes, since it can affect downstream applications. If LiCl is not necessary, why not try NH4Ac? I have found it to be a very reliable salt for RNA precipitation with little effect on downstream applications.

Best of Luck.




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