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what solution in upper layer of PCI solution?


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#1 koreanraichu

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Posted 20 January 2019 - 07:01 PM

Greetings.

 

I hard to explain so drew it.

IMG_8092.JPG

 

As you know, PCI solution(phenol:chloroform:Isoamylalcohol 25:24:1) have a two layers. PCI is lower layer, and there are solution located upper.

what's this? I founded about this, someone said Tris(50mM, 10mM, 100mM) or Isoamylalcohol, but I don't agree Isoamylalcohol because phenol and chloroform, Isoamylalcohol can mixed because phenol and chloroform are organic solvent, and ethanol can mix with organic solvent. (and Isoamylalcohol have non-polar parts)

 

Then My question Is Two:

1. what solution in upper layer of Phenol:chloroform:isoamylalcohol 25:24:1?

2. if that solution is Tris, It role just prevent oxidation of phenol? never role of buffering agent? or it works by buffering agent for prevent oxidation of phenol?



#2 bob1

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Posted 20 January 2019 - 08:10 PM

This is either buffered Tris or water used to keep the PCI at the correct pH for the experiment you are using - 7.4 for DNA and 6.3 (IIRC) for RNA. The water promotes an acidic environment that means that DNA is excluded from the aqueous phase during the extraction. Tris buffer keeps the pH at around 7 which means that DNA is preferentially sequestered in the aqueous phase.






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