What is the difference in using phenol:chloroform:isoamyl alcohol at 25:24:1 v/v versus just using chloroform:isoamyl alcohol at 24:1 v/v?
I need to extract some DNA but seems there is only the phenol:chloroform:isoamyl alcohol left but the protocol just calls for chloroform:isoamyl alcohol so I am wondering what the difference would be?
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dna extraction question
Started by claritylight, May 09 2011 05:29 AM
2 replies to this topic
#1
Posted 09 May 2011 - 05:29 AM
#2
Posted 09 May 2011 - 07:12 AM
Chloroform helps to denature proteins as well as removing residual phenol (since phenol is not very soluble in chloroform) ...so it is not a really good idea to use phenol:chloroform instead.
You can try to find some chloroform without isoamylalcohol ...that would do the trick as well. The isoamylalcohol is not that essential.
Regards,
p
You can try to find some chloroform without isoamylalcohol ...that would do the trick as well. The isoamylalcohol is not that essential.
Regards,
p
#3
Posted 10 May 2011 - 05:29 AM
In addition to denaturing protein, phenol also acts as a buffer and is used in RNA isolations to keep the pH acidic. Acidic phenol:chloroform is used for RNA isolation; at this pH DNA will be seperated into the interphase and RNA will be left in the aqueous phase. If the pH of your phenol:chloroform is below 7, you will lose DNA. I believe the isoamyl alcohol acts as a defoaming agent, probably allows the phases to mix more completely and efficiently.