UCSF

Extract DNA from Paraffin Blocks

Day 1

  1. Cut 10-20X (30um) sections of formalin fixed paraffin samples into Eppendorf tubes.
  2. Add 1ml Xylene and incubate at RT for 15min.
  3. Spin down for 5 min at 13000 rpm and discard supernatant.
  4. Add 1ml of 100% ethanol and incubate at RT for 15 min.
  5. Spin down for 5 min at 13000 rpm and discard supernatant.
  6. Add 500ul of proteinase K buffer (50mM Tris pH 8, 1mM EDTA, 0.5% Tween 20).
  7. Incubate overnight at 55°C shaker.

Day 2–5

  1. Add 20ul proteinase K (stock solution 20mg/ml in water, store at -20°C) Final Conc.=0.4mg/ml
  2. Incubate overnight at 55°C shaker. (Solution will become clear. You may increase proteinase K conc. up to 1 mg/ml)

Day 6

  1. Add 500ul Phenol Chloroform into tube and wait for 5 min at RT.
  2. Spin down at 5 min with 13000 rpm.
  3. Get off the upper layer. (we need the upper layer)
  4. Mix it with 500ul of PCI in Eppendorf tubes. (Phenol-chloroform-isoamylalcohol extraction 1:1 v/v)
  5. Shake gently and incubate for 10 min at RT.
  6. Spin down at 5 min with 13000 rpm.
  7. Collect the supernatant into new Eppendorf tubes.
  8. Add 300ul of 7.5M ammonium acetate, 1ml cold 100% ethanol and 5ul glycogen(stock (stock = 20ug/ml)
  9. Shake gently and incubate at -20°C for 2 hours or overnight.
  10. Spin down for 30min at 13000 rpm and discard the supernatant.
  11. Air dry pellet and dissolve in 25ul of water or TE buffer.
Proteinase K buffer

Tris pH=8

0.5 ml

0.5M EDTA

40 ul

10% Tween

1 ml

dd H2O

28.5 ml

Total

20 ml


Proteinase K dissolve solution

Tris HCL pH=7.5

0.1 ml

CaCl

0.2 ml

Glycerol

10 ml

Water

10 ml