Wednesday, June 01, 2005

Description
Small scale chromosomal DNA isolation from brain tissue. Suitable for PCR amplification.

Procedure
1. Cut +0,25 g brain tissue (best fresh of freshly frozen tissue) in smaller pieces.
2. Add 850ul Lysing buffer (100mM NaCl; 10mM Tris-Cl pH 8; 25mM EDTA; 0.5% SDS; 0.1mg/ml ProtK; 1mg/ml RNAse).
3. Homogenise tissue.
4. Incubate overnight (>3 hours) at 50 oC while gently rocking.
5. Spin 2 min 1000rpm in Eppendorf.
6. Take 700ul of the supernatant.
7. Add 1 volume of phenol:chloroform (1:1).
8. Spin 10 min 3000rpm.
9. Take the supernatant and add again 1 volume phenol:chloroform (1:1).
10. Spin 10 min 3000 rpm.
11. Take supernatant and precipitate DNA by adding 1/10 volume NaAc pH 4.8 and 2 volumes of ethanol (100%).
12. Gently mix and spin 10 min 13000rpm.
13. Suspend the pellet in 250ul SuperQ water (or TE if required).

Approximate yield will be about 60ug of chromosomal DNA of reasonable quality (more than clean enough to perform PCR).


Recipes
see above

Supplies
none

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