isolation of DNA from mouse tails
ttha at uhura.cc.rochester.edu
Sat Jul 20 09:16:26 EST 1996
In <4sopcf$cdh at fremont.ohsu.edu> "John J. Shaskus" <shaskusj at ohsu.edu> writes:
>I've been having problems with a DNA isolation procedure for mouse tails
>and would welcome suggestions to alter or replace it.
>The procedure entails:
> 1) Digestion with proteinase K
> 2) Addition of NaCl to 1.5M
> 3) Extraction with CHCl3/Isoamyl alcohol
> 4) Precipitation with EtOH
>The problem: Upon addition of EtOH, in addition to DNA precipitation,
>the solution clouds up. Over time a H2O-miscible oil precipitates (i.e.
>it's not CHCl3). The oil makes it difficult to remove the DNA and seems
>to inhibit restriction enzymes to some degree.
>shaskusj at ohsu.edu
Our protocol in short form is this:
Tail lysis buffer is Tris/SDS/EDTA
Add proteinase K, digest overnight 55C
Add RNAse A, digest 2 hrs 37C
Extract once with equal volume phenol
Extract twice with equal volume phenol/chloroform/isoamyl alcohol 25/24/1
Extract once Chloroform/Isoamyl alcohol 24/1
PPT with equal volume 2-propanol
Remove DNA pellet with pipetter (200 ul with tip cut off)
Transfer pellet to 75% ethanol(washes out salts)
Transfer pellet to 100% ethanol
Decant ethanol and air dry
Add 100 ul TE, solubilize overnight at 37C.
Tom Thatcher | You can give a PC to a Homo habilis,
University of Rochester Cancer Center | and he'll use it, but he'll use it
ttha at uhura.cc.rochester.edu | to crack nuts.
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