DNA from paraffin tissue section.

[XIAO-MIN SU]

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