phenol-chloroform-extraction - (Sep/26/2005 )

Hi,

on DNA result from plant DNA extraction. i get RNA.
it is caused from PCI? different pH of phenol?
thanks

tomoe

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You may like to try AquaPlasmid and AquaGenomic, which do not use phenol/chlorform at all for DNA extraction.

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Dear all,

anyone who knows how to prepare the buffer 5.1 for RNA extraction? Does that all the reagent need to be RNase free? Where can we get them?

Thanks a lot

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JUST A NEWBIE QUESTION.
IN PHENOL-CHLOROFORM DNA EXTRACTION WE USE PHENOL,BUT IN LIQUID CONDITION.
I ORDERED PHENOL BUT THE ARRIVED ONE WAS IN CRYSTAL CONDITION (MERCK).
I CANT FIND ANY INFO ABOUT THAT.

HOW SHOULD I PREPARE LIQUID-PHENOL FROM CRYSTAL FORM.

THX.

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you can dissolve 500g crystal phenol in 500 ml 1M tris pH 8.0 (if you want phenol solution for DNA extraction) or 50 mM Sodium acetate pH 4.0 (when prepare acid Phenol for RNA extraction). Keep in mind check pH of phenol solution before use and discard this solution when phenol turns red/brown.

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back to the main topic about phenol/chloroform extraction....if I have purified PCR product, which is purified by the gel from all the taq and dNTPs I suppose....I go on and digest it and then diactivate the enzymes.....molecular cloning book says phenol/chloroform after that and then ligate .. but why to if DNA is already purified? that will only lead to more loss.....

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I used to melt the crystalline phenol in a 37C water bath, then add (in the hood) an equal volume of Tris-Cl, pH8 and mix to saturate the phenol. After stabilization of the mix, pull off most of the top layer of excess Tris buffer and add a volume of Chloroform:Isoamyl Alcohol (24:1) equal to the original volume of Phenol (so for 500 mL phenol, use a mix of 480 mL Chloroform + 20 mL IAA. The IAA cuts down on the Chloroform tendency to foam when mixed). You'll need a larger container for this mix - anything screw-capped is fine (I used a 2L screw-top Erlenmeyer), as long as the final mix is stored in one or more brown jars/bottles (light-sensitive contents). The best way to do this is to decant the phenol-Tris mixture into the larger container, then carefully wash out the original phenol jar with the Cl:IAA mixture several times, pouring off each time into the larger container. There are some caveats:
1. This stuff is VERY nasty - especially when the caustic Phenol is mixed with the fat-solubilizing Chloroform. DON'T spill it on exposed skin. Wear lots of protective gear (goggles, rubber apron, etc.)
2. Phenol itself is slippery; mixed with Cl:IAA, it becomes very slippery indeed, and has a nasty habit of sneaking out under jar lids when the jar is shaken. This may lead to dropping the large jar of P:Cl:IAA, with predictably unpleasant (and highly dangerous) consequences for humans and lab equipment. Wear very "grippy" gloves - Rubbermaid dish-washing gloves worked for me, over the standard latex or nitrile lab gloves.
3. Liquid P:C:IAA can be stored at 4C for some time (up to months), but should not be used when the normal faint yellowish darkens or turns pinkish.
I know this is way more than you need - but I've found over the years that too much information is almost always better than not enough...

Oh, and Kathy - it's a good idea to clean up the enzyme/salt stuff out of your mix before ligating. You don't always have to phenol extract it, though - there are a number of good column-based cleaning kits out there from Qiagen, Promega etc. They're quicker, easier, less unpleasant, and have a smaller loss.

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Hi,
another question to this topic:
Which is the "best" method (ie most high quality DNA yield) if available samples contain only little DNA amounts (eg tiny insect eggs or legs). Chloroform/Phenol, Salting-out, Kit, or...??
Thanks
hobglobin

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that means that if i want to extract the DNA form adenovirus lysate with phenol-chloroform (1:1) i have to prepare the phenol solution as you said (500g crystal pheno in 500 ml 1M tris pH 8) and mix it with chloroform???

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See here:

http://openwetware.org/wiki/Phenol

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