IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

DNA extraction from single paraffin sections

aforus aforus at radium.uio.no
Fri Nov 3 09:21:52 EST 1995

In article <472go9$7u8_001 at leeds.ac.uk>, PATJAR at leeds.ac.uk (J.A.
Randerson) wrote:

> Hi 
> 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!
> Thanks 
> 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

More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net