DNA extraction from wax blocks

It is important to note that the paraffin-embedded specimens will be fragmented due to formalin fixation and embedding with hot wax, and the extracted DNA fragments will be fragmented, and it is difficult to store them for a long time. It is best to amplify or do the corresponding immediately after extraction In the experiment, when stored at -20 degrees for more than a week, the DNA will be degraded obviously, and it will not be able to do anything in a long time.

1 Put three 10um thick sections of paraffin-embedded tissue into an EP tube.
2 Add 1ml of xylene to each tube, cover tightly, let stand for 30 minutes, centrifuge at 12000rpm for 15 minutes, suck and discard xylene, and save the sediment.
3 Repeat step 2 twice.
4 0.5ml absolute ethanol, invert and mix the sediment for 5 minutes, centrifuge at 12000rpm for 15 minutes, suck away and discard the absolute ethanol, and save the sediment.
5 Repeat step 4 once.
6 Vacuum dry ethanol or air dry.
7 Add 200ul of digestion solution (final concentration Tris-Hcl 0.05mM, EDTA0.o1mM, Tween20 0.5%, protease k100ug / ul) to the dry sample, mix the sediment and digestion solution, 55 degree water bath for 3 hours or 37 degree overnight It is best to have a shaker during this period, or shake it manually every 10 minutes.
8 100 degrees for 10 minutes, inactivate proteinase k.
9 Briefly centrifuge to settle the moisture on the tube wall and cap.
10 Add the same volume of redistilled phenol as the digestive juice, and invert gently for 5 minutes. Centrifuge at 4000rpm for 5 minutes, carefully draw the supernatant, and discard the middle white precipitate and the lower supernatant (the white precipitate must not be sucked up, it is very important!)
11 Add half volume of re-distilled phenol in digestion solution, half volume of 24: 1 chloroform: isoamyl alcohol, and invert gently for 5 minutes. Centrifuge at 4000rpm for 5 minutes, carefully draw the supernatant, and discard the intermediate white precipitate and the lower supernatant (the white precipitate treatment is the same as step 10!)
12 Add an equal volume of 24: 1 chloroform: isoamyl alcohol to the digestive juice and invert gently for 5 minutes. Centrifuge at 4000rpm for 5 minutes, carefully draw the supernatant, and discard the intermediate white precipitate and the lower supernatant (the white precipitate treatment is the same as step 10!)
13 Add 2 volumes of ice-cold absolute ethanol (pre-cooled at -20 degrees for half an hour) to the supernatant, and precipitate for more than 1 hour.
14 Centrifuge at 12000rpm at 4 degrees for 15 minutes, discard ethanol, vacuum dry, and dissolve the precipitate with TE30ul.

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