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

hey everyone

this is a new question
i am currently doing a project at uni where my plasmid dna has and been through restriction, polishing and ligation of the insert. now i need to extract the plasmid dna fromthe solution and i was wondering is the phenol-ethanol extraction procedure at pH 8 explained earlier suitable as i will need the recombinated dna for real time pcr.

thanx

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How could you equilibrate your phenol.... I use the protocol on Molecular Cloning (Sambrook, 2001)...But as far as I can go is pH7.0 > pH 7.5 ... I have never been able to make my phenol reach pH 8.0..... any suggestion please!

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[quote name='LLL1969' date='May 30 2006, 03:10 AM' post='53475']
[quote name='fred_33' post='28508' date='Oct 20 2005, 05:03 AM']
well i assume alcool is a kind of stabilizant... so IAA or octanol...if it works, go.

phenol pH 8 : it's phenol solution saturated with Tris pH 8
[/quote]



From what I know IAA is added to prevent foaming not to stabilize

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Hi!everyone,

I am using SDS-phenol method to isolate RNA, but my RNA products are always contaminated by DNA. I have specially adjusted the pH of the aqueous phase to an acid phase during phenol/chloroforme extraction. I have seen someone suggested an improvent method to reduce DNA contamination during RNA extraction( which is discribed bellow), does it work? I want to know the mechanism that it works. I also want to know if the remnants of the denaturated cells coprecipitated with RNA pellet? Where is the DNA? Does it remian in the aqueous phase?
The Suggested protocol is discribed as:
There is a modification of the guanidinium thiocianate-phenol method (trizol extraction) which reduces genomic DNA contamintion: Nucleic Acid Research, 1993m vol. 21 N 8 2019-2020. The modification involves the addition of a brief RNA selective precipitation step following the lysis of the cells with the guanidinium thiocianate solution. This is achieved by the addition of 1/3 volumen of 95% ethanol and incubation of the mixture on ice for 5 minutes. Following centrifugation for 15 minutes the supernatant is removed and the RNA pellet dissolved in 1/2 of the original volume of guanidinium thiocianate solution

Thanks in advance!

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In my hands It seemed that the acid pH of the phenol solution can not effectively eliminate the DNA. I use SDS-PHENOL to extract RNA, and an equal volume of phenol/chloroform is used. Others suggested use a volume ratio of 5/1 of phonol/chloroform. If anyone know it does work ,please tell me.

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Heat it at 60℃, and balance it with tris buffer at ph 8.0

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The phenol should be water saturated for isolating RNA! I highly recommend that you buy it but not prepare it yourself.

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The thing with phenol/chloroform is that although it's a great extraction, you always lose some sample bearing material as you can't (don't want to try to) recover all of the aqueous phase. In samples that contain minimal amounts of DNA this could lead to excessively high losses.

I suppose, if you were really careful and used minimal extraction volumes and then concentrated all of your extract via a Microcon filter (or if you're confident of your technique 70% ethanol wash followed by 100% ethanol wash) then you could hope to recover a high amount of what's in there.

I couldn't guess at percentage recovery, though. It depends on too many local factors.

Your best bet, would be some sort of lysis of the DNA bearing matrix and then total digestion of proteins. Then concentrate the DNA via a microcon unit (I've successfully used these units to concentrate 1 virus out of a litre of sea water with 100% recovery).

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hi....
you have to destilate phenol crystal. destilation, it may works!
thx

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New question. For DNA extraction (keeping it in the aqueous phase), one is suppose to use phenol-chloroform saturated with Tris pH 8. What about pH higher than 8? Will this have any negative consequences?

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