Crude Lysis and other DNA Extraction Methods

Other DNA extraction methods

• Limiting specimens (fixed tissue, dried blood, bone).

• Rapid extraction for routine testing.

Crude Lysis (4)

• DNA does NOT need to be pure.

• RE digestion, gel electrophoresis, screening large amounts of DNA, some PCR, challenging specimens.

• Proteolytic enzymes - Proteinase K (PK)

• Chelating resins – Chelex

Proteolytic enzymes

Proteinase K (PK)

• Chews up muscle

Chelating resins

• Chelex

• Chelex removes multivalent cations

  • DNAses (+) destroys remaining DNA.

    • When we use resin, the polyvalent cation binds with magnesium ion, so it stops DNA from being destroyed.

• 300 mL 5-20% chelex resin

• High yeild but not pure DNA

Fixed tissue

• 1 micron = 1X10^-7 meter or 0.0001 cm

• Thin slice of tissue

Fixed tissue steps

1. ~20 micron

   sections

2. Macrodissect or

   microdissect

3. Digest

   (proteinase K,

   Tris buffer)

Whatman FTA paper

• Good way to transfer sample when they need to go to another lab

• You can take a punch from the paper to use sample for testing

• Uses Lysis buffer