isolation of DNA from mouse tails

[Tom Thatcher]

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.
>Thank you

>John Shaskus
>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.