song.65 at OSU.EDU wrote in message <36E98669.A53C510F at osu.edu>...
>My name is Nirav Shah, student at the Ohio State University.
>I need help with DNA extraction from paraffin blocks.
>I have tried this protocol but it didn't yield any products.
>Anyone that can help me please e-mail me at shah.198 at osu.edu>>Here is the protocol I used:
>1 Scrape off area of interest with a sterile (#11) scalpel blade and
>transfer paraffin shavings
>to sterile 0.5 ml tube.
>2 Rinse shavings from blade with n-octane washes until there is 0.5 ml
>in centrifuge tube.
>3 Vortex for 20 minutes
>4 Centrifuge for 5 minutes @ 6000 rpm
>5 Pipette off n-octane using a pipetman with micro-capillary tip.
>Usually, paraffin sticks to
>sides of tube, avoid sucking it up.
>6 Add 0.5 ml 100% ethanol to tube.
>7 Vortex for 20 minutes
>8 Centrifuge for 5 minutes @ 6000 rpm
>9 Pipette off ethanol. Avoid paraffin, which should be in a pellet at
>10 Evaporate ethanol until pellet is dry several hours in a sterile
>11 Add 100ul digestion buffer (50mM Tris pH 8.5, 1mM EDTA, 0.5% Tween
>20) and 2ul
>Proteinase K stock solution (20 mg/ml in sterile H2O).
>12 Place the sample tubes in a floating rack in a 55 C water bath.
>Incubate for 48 hours.
>13 Spin tubes briefly, then heat tubes at 95 C for 10 min to inactivate
>14 Centrifuge at 6000 rpm for 1 min to pellet undigested material.
>Again the e-mail address is shah.198 at osu.edu>>
This seems like a very long method which could be done quicker. We extract
DNA from sectioned paraffin blocks by dewaxing first in Xylene (2 X 5
minutes), 100% ethanol rinse (2 X 1 minute), 95% ethanol (1 minute), leave
to air dry (5 minutes). Scrape tissue off with sterile syringe needle or
scalpel blade using 50 ul lysis buffer (0.04% Proteinase K, 10 mM Tris-HCl
(pH 8.0), 1 mM EDTA and 1% Tween-20). Incubate overnight at 42 C. The
following day inactivate at 95 C for 10 minutes, then store at -20 C. 4-5 ul
should then be used in your PCR. We use this method to amplify <200 cells
from paraffin tissue so you shouldn't need a lot of tissue. We can also
amplify >300 bp products using this method. If you have problems with this
it may be your PCR reaction that is going wrong (i assume that is what you
are using the DNA for).
Roy Castle International Centre for Lung Cancer Research