Tissue collection, storage, microdissection, sectioning: See separate protocol.
Tissue handling: Note that all fresh tissue should be handled as BioSafety Level 2 materials (wear gloves, lab coat, etc.).
DNA extraction: The following protocol is based on a standard phenol DNA extraction protocol. Other protocols, and versions of this protocol, are also acceptable.
1. Take pre-cut samples out of the –80°C freezer and thaw.
2. Add 10 ml of PBS (Ca-Mg Free) in hood in BSL2 room to dissolve OCT. Invert tube to make sure all of tissue is in the solution and not stuck on the tube walls.
3. Spin down tissue 1400 rpm (500 x g) for 5min.
4. Remove supernatant carefully watching the tissue pellet.
Note: Repeat steps 2-4 (with 5mls PBS) if it looks like there is significant “sticky” OCT left in the tube. If you are repeating the PBS wash step you do not have to get too close to the tissue pellet the first time.
5. Resuspend pellet by vortexing. Add 950 ul digestion buffer (100 ul 10X PCR buffer [100 mM TRIS, 15 mM MgCl2, 500 mM KCl), 5 ul 0.5% tween 20, 845 ul H2O) and 30-50 ul of 20 mg/ml Proteinase K (PK, Sigma P2308).
Note: this volume should vary depending on the size of the tissue pellet. If the pellet is bigger then add 2 ml total of buffer + PK.
6. Resuspend pellet by vortexing, and pipeting up and down and place in a shaking 50°C water bath overnight. The next day make sure all the tissue has been digested. If necessary, add more PK buffer and allow to digest for a few more hours.
7. Split each tube into two 1.5ml tubes (500 ul per tube).
8. Add 500ul (or equal volume) of room temp Phenol/Chloroform/Isoamyl Alcohol (PCI from Amaresco) into each tube and vortex for 10sec.
Note: PCI is the lower organic layer.
9. Centrifuge at 14000 rpm (max) for 2 min at room temp.
10. Remove aqueous layer into a new tube and repeat the PCI extraction (steps 8-10).
11. Aliquot the aqueous phase into as few 1.5 ml epindorf tubes as possible. Maximum volume per tube is 350 uls. Add 1/2 volume of 7.5 M ammonium acetate and mix.
12. Add 2.5 X 100% ethanol, mix by inversion. Leave at RT for 2 hrs, or ON at –20°C.
13. Centrifuge at 14000 rpm for 15 min @ 4°C.
14. Decant supernatant immediately.***Note watch for the pellet***
15. Wash pellet in 70 ul of 100% ethanol. Make sure you rinse sides, rim of tube. Spin at 14000 rpm for 5 min and dump supernatant.
16. Blot with Kimwipe. Air dry pellet.
17. Add 20-50 ul of TE. The volume will depend on the pellet. (avg ~ 30 ul)
18. Leave at RT for 2 hrs or ON.
19. Place tubes into 65°C for 1 hour (to inactivate DNAse).
20. Combine tubes. Rinse out “empty” tubes with 20 ul TE (the same 20 ul can be used to rinse out all tubes).
21. Measure DNA concentration using a fluorometer with a known standard DNA solution. Very small amounts of DNA can be quantitated by TaqMan analysis with a standard assay.
22. Store DNA at 4°C for short periods, or colder for longer periods. Repeated freezing and thawing may lead to shearing of DNA into smaller pieces.
