In article <472go9$7u8_001 at leeds.ac.uk>, PATJAR at leeds.ac.uk (J.A.
>> Can anyone tell me of a way to extract PCR quality DNA from a single
> paraffin section. I have tried a number of methods including standard
> protinase K with phenol/chloroform extraction but to no avail.
>> Email to patjar at leeds.ac.uk if you can help. I'm getting desparate!
> Juliette Randerson, University of Leeds
You may want to try the following procedure:
Prepare sections (>10 micron) by slicing. Minimum 10 (you could take more
if possible). If possible, remove excess paraffin.Cut the tissue into very
small pieces with a surgical blade and transfer to a 50ml tube (The pieces
may be a little ³static electric² - so be careful that you get them all
into the tube).
Add 5 ml of lysis buffer: 10 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA. Place
at -70 °C for 30 min (may not be necessary; we normally do this for frozen
Thaw, then add 5 ml of lysis buffer with 2% sarcosyl and 50-100 mg/ml
proteinase K. Set to rotate at 65 °C overnight. The parraffin melt and
stay molten at this temperature, and proteinase K is more effective.
Phenol for extraction should be saturated with 0.5 M Tris, and pH should
be between 6 and 7. Keep the phenol at 65 °C.
Add an equal volume (10 ml) of phenol to the 65 °C lysis suspension, leave
to rotate at 37 °C for 30 min. Remove the phenol, add 10 ml of 1:1
phenol:chloroform, set to rotate at room temperature for 30 min. Remove
lower (organic) phace, add 10 ml of chloroform, let rotate at room
Move upper phace to a 30 ml sentrifuge tube. Add 1/10 of the volume (1 ml)
of 3 M NaAcetate, then 10 ml of isopropanol. Mix gently. Add another 10 ml
of isopropanol, and leave to precipitate at -20 °C ( 30 min or overnight).
Spin 60 min at 10 000 rpm , 4 °C. DNA will pellet. Wash the pellet once
with 75 % ethanol, spin 30 min at 10 000 rpm, 4 °C. Use a pipette to
remove as much ethanol as possible, the rest is removed by evaporation (37
°C). Dissolve the pellet in TE (10 mM Tris pH 7.6, 1 mM EDTA).
I haven´t tried PCR with this DNA, but it works very well for CGH (which I
should think requires higher quality of DNA). Good luck.
Anne Forus, Dept. Tumor Biology, The Norwegian Radium Hospital, Oslo