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Phenol extraction


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21 replies to this topic

#16 HomeBrew

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Posted 20 July 2010 - 04:14 AM

I think the problem you're having is due to using straight phenol, rather than buffer-saturated phenol. Prepare buffer-saturated phenol as shown under "Preparation of TE buffered Phenol" here.

#17 Sluvah

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Posted 20 July 2010 - 04:31 AM

I'm sorry, I forgot to precise. It's buffered saturated phenol, pH=8

#18 Maddie

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Posted 20 July 2010 - 05:31 AM

I usually decide how many phenol-chloroform to do after looking at the first one: how is the interphase? Is the aqueous phase cloudy? Usually, 2 are plenty enough. I'm also surprised to see so many steps in your protocol.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#19 Sluvah

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Posted 20 July 2010 - 05:48 AM

My boss is cautious. REALLY cautious....

Fortunately, my boss is on vacation. So I will be able to freely adjust the protocol... Because the more I read about DNA extraction, the more I think just P/C + C extraction should be enough.

Thanks everyone !

#20 Sluvah

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Posted 23 July 2010 - 06:06 AM

Hello,

Just to let you know,

I made a test with P, P\C then C extraction of a smaller culture volume with success. I think the error was too high concentration of detergent (SDS and sarcosyl) and a too low centrifugation speed (4000RPM, I haven't been able to find the rotor radius, so I can't convert this speed into "g". For a bigger volume, I will use another centrifuge where I can switch RPM to RCF)

My only problem is that I ressuspend my DNA pellet into too much TE buffer : It's too diluted, but I will re-precipitate it as soon as I can. I still need to check the absence of phenol residue, but I'm confident.

Thanks every one !

#21 Maddie

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Posted 23 July 2010 - 07:18 AM

To remove phenol residues, you can do PCI followed by a butanol extraction.
Good luck and good job.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#22 Flopilus

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Posted 03 April 2012 - 08:03 AM

How can I prepare a 38% bufer-saturated phenol (v/v) solution? I need it for an Extraction buffer to isolate RNA.

Thanks.




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