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Why isnt Phenol miscible in Tris Buffer?


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#1 lolcakes

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Posted 07 September 2010 - 03:03 AM

hi everyone,

i was wondering why phenol is not miscible in the tris buffer during genomic DNA isolation? its at the bottom layer undernearth the tris buffer phase.
give me 2 reasons why these two cannot be mixed? (used phenol/cholorform/isoamyl alcohol (25/24/1))

#2 donny

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Posted 07 September 2010 - 06:17 AM

hi everyone,

i was wondering why phenol is not miscible in the tris buffer during genomic DNA isolation? its at the bottom layer undernearth the tris buffer phase.
give me 2 reasons why these two cannot be mixed? (used phenol/cholorform/isoamyl alcohol (25/24/1))


Tris has a pKa~8 while phenol has a pKa~10. Therefore, phenol is not acidic enough to be deprotonated by tris to dissolve in the aqueous phase. Since phenol is predominantly in the uncharged form, it is way more soluble in the bottom organic phase (chloroform is denser than water).




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