On Tue, 8 Oct 1996, Gregory Jacobson wrote:
>> I need get some DNA from part of a tissue section (Breast Ductal
> Carcinoma) for PCR and subsequent CFLP or SSCP. I have removed the
> paraffin from the sections using xylene and alcohol washes. I will use a
> silcon pen to isolate the tumour cells.
>> I would be grateful for advice about a method for protease treating the
> tissue :
> Is TE the best buffer to use?
> How do I apply the proteinase K? (With buffer? or after soaking tissue in TE?)
> Should I incubate the slide at 42 degrees C? (higher?)
> Can I simply pipette the DNA off the tissue section after proteinase K
> treatment?
>> Thanking you in advance,
> Gregory Jacobson
> 2nd year MSc
> Uni. of Waikato
> Hamilton
> New Zealand
>> Please email : gmj at waikato.ac.nz>>Gregory,
Here is the protocal we use.
Scrape the tissue off the slide. Transfer into a 1.5 ml microfuge tube.
Spin the tissue to the bottom of the tube.
For a 6 micrometer thick, and 5 mm X 5 mm tissue, add 50 microliter 1 X
TK buffer. Vortex for 5 sec. Bump down in microfuge. Incubate at 56 C
overnight.
Spin down briefly. Add 100 microliter 5% chelex 100 in 0.1 TE9. Vortex
for 5 sec. Bump down in microfuge. Incubate at 100 C for 10 min. Vortex
for 5 sec. Spin in microfuge for 2 min. Store at -20 C.
The DNA now can be used directly as PCR template. We use 1 microliter in
a 10 microliter reaction.
10 X TK buffer:
50 microliter Tween 20
20 microliter 100 mg/ml proteinase K in TE9
930 microliter TE9
TE9:
121 g Trizma base
14.9 g EDTA
5 ml 4M NaCl
Add water to 2 liters
Adjust pH to 8.9
Good luck!
Xiao-min Su
Johns Hopkins University
Dept. of Pathology
