Ethanol precipitation problem - (Apr/25/2006 )

Hi,

I am a biotech student, and after two months of struggling to get a ligation & transformation to work, have discovered that I have been losing my DNA during ethanol precipitation, which is the method commonly used in the lab I am working in.

Does anyone have any tips/advice on what I could be doing wrong?

Any assistance would be greatly appreciated

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What is the mass of DNA you are trying to precipitate??
If too little, you may need a carrier like glycogen or acrylamide.
What is the salt you are using?
0.1x vol 3M Na Acetate pH 5.2 is typical
Are you using 2x vol 95% etoh?

Put the DNA (after adding salt, vortex and ethanol, rock back and forth 10 times) into the -20 for an hour or so. Spin 14kxg 15min 4C. If you have a reasonable amount of DNA (or carrier) you should see the pellet.

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How large is the insert? If it's small (say 100 - 200 bp), you might want to use ammonium acetate, rather than sodium acetate. It's been a while since I did any, so you should check up on the concentration. Try this link
http://www.biochem.ucl.ac.uk/bsm/nmr/proto...ls/dnaprep.html

Please note: I just pulled this off Google, so I make no guarantees, but it looks about right. :-)

Good luck!!

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Thanks for that, I will give it a shot.

I am not sure of the mass of DNA - it has been cut out of an agarose gel and purified.

I have been adding 5 microlitres of 5M NaCl to 100 microlitre sample, adding 2xvolume of 100% ethanol, putting at -20 for around half an hour, spinning at 15k for 10min at room temp, removing ethanol, then adding 2xvol 70% ethanol. Then, spin again for 10min at 15k, remove all ethanol and dry at 65 degrees on a heating block.

Is it important to centrifuge at 4 degrees? I only have access to a centrifuge at room temp.

Thanks again, I will give your method a go today

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My insert is 840bp so I think that NaCl should be ok?

THanks anyway

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Hi,

I tried the precipitation again yesterday and this time followed your advice as much as possible. The problem is that I do not see a pellet in the tube and, as these are my first attempts at precipitation, I guess I am losing it somewhere along the way.

Someone in my lab recommended trying the ligation and skipping the precipitation step. As I am doing purification first, I could try eluting the DNA in a 30 microlitre volume instead of 50 and try to ligate with that.

Do you think this is worthwhile? I realise that this will decrease the chances of success because of a higher volume, but at the moment I am losing my DNA before I even get to the ligation step...

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Did you really mean NaCl? I've only heard of using acetate, never chloride. At 840 bp, either type of acetate should do.

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hi
acetate is ok. try 10vol buthanol. Doesn't need any added salts for precipitating with high efficiency and quick.
10 vol of butanol, vortex well to homogeneize spin max speed 10'. pellet is ok to ethanol wash.

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