DNA extraction from paraffin-embedded tissu
Posted 27 November 2003 - 05:32 AM
- how long I have to put my simple in Xylene?
- Is it necessary to re-hydrate the tissu'simple?
Or better, there is a protocol on line, what are the links?
thanks for your help!
Posted 28 November 2003 - 02:02 AM
1. Cut 10-20X 10 um sections of formalin fixed paraffin samples into eppendorf tubes.
2. Add 1 ml xylene, mix, incubate at 55 C for 15 mins. Release pressure, spin down for 2 minutes in eppendorf ultramicrofuge, pipet off & discard supernatent.
3. Repeat Step 2.
4. Add 100% ethanol, incubate for 15 mins. Spin down.
5. Remove ethanol, Repeat Step 4, allow pellet to air dry.
6. Add up to 1 ml of Proteinase K in digestion buffer to a final concentration of 0.3-0.5 mg/ml (less buffer for smaller tumors).
7. Incubate overnight, shaking, at 55 C.
8. Add fresh concentrated PK (same amount as day 1 from 20 mg/ml stock).
9. Incubate overnight at 55 C.
10. Repeat step 8 & 9.
11. Add equal volume PCI to the PK digested aqueous solution, spin 2 mins, remove upper aqueoues phase to new tube. (repeat PCI extraction if necessary -- it usually is).
12. Take 330 ul aqueous phase per eppendorf tube, add 1/2 original volume (165ul) ammonium acetate (7.5M) [can add 1-3 ul Glycogen here to preserve low amounts of DNA. Glycogen makes a nice visible pellet] and 2-2.5X volume with 100% ethanol. Let sit at RT for ~2 hours or overnight at -20 C.(better O.N.).
13. Spin for 20 minutes at 4 C, decant ethanol (rinse pellet with etoh if it doesn't move), blot dry, air dry.
14. Carefully resuspend the pellets in small volumes of TE buffer (~15-20 ul) and let dissolve at RT overnight (or 55 C for 2 hours). Combine tubes from the same sample.
15. Measure DNA concentration on the Fluorometer. Ideal DNA concentration is from 200-500 ug/ml.
16. Denature the DNA at 70 C before running 0.2 ug on 1% gel to check the size. Size should range from 100bp-3Kb.
Digestion buffer: 100 mM NaCl/10mM Tris-HCl, pH 8.0 and 25 mM EDTA, pH 8.0/0.5% SDS. Store at RT.
Proteinase K: Stored as 20 mg/ml aliquots at -20 C; Can be refrozen a few times. Use Proteinase K at 0.3-0.5 mg/ml in digestion buffer
PCI: phenol/chloroform/isoamyl alcohol (from Ameresco)
Posted 28 November 2003 - 10:53 AM
1. Use a small section (not more than 25mg) of paraffin-embedded tissue
2. Add 1200 ul xylene, vortex vigorously.
3. Centrifuge at full speed for 5 min
4. Remove supernatant
5. Add 1200 ul 100% ethanol and mix by vortexing
6. Centrifuge at full speed for 5 min
7. Remove ethanol by pipetting
8. Repeat 5-7 once
9. Dry at 37C for 15 min