Worm Lysis and DNA Isolation

This protocol can be used to isolate genomic DNA from one plate of worms.

Procedure

1. Wash worms off plate with 1 mL distilled and autoclaved water or M9 solution, transfer to centrifuge tube
2. Spin down or allow worms to settle to bottom of tube

   Optional further steps:

   1. Remove supernatent
   2. Wash with 1 mL distilled and autoclaved water to remove bacteria
   3. Spin down or allow worms to settle to bottom of tube, remove supernatent

1. Prepare PCR tube with 40 uL of lysis buffer (see below) including proteinase K
2. Add 10 uL of worms from the pellet in the centrifuge tube to the prepared PCR tube
3. Optional: Put PCR tube into -80 freezer for 15-30 minutes to crack/weaken worm cuticles
4. Incubate at 65C for 2-4 hours
5. Incubate at 95C for 15 minutes (to inactivate the proteinase K)
6. Store at -20C (this is the isolated DNA)

Worm Lysis Buffer

Concentrations:

50 mM KCl
10 mM Tris pH 8.3
25 mM MgCl2
0.45% NP-40
0.45% Tween-20
0.01% gelatin
To make 50 mL:

2.5 mL of 1 M KCl
0.5 mL 1M Tris pH 8.3
0.25 mL 0.5 M MgCl2
5 mL 4.5% NP-40
1.125 mL 20% Tween-20
5 mL 0.1% gelatin
Proteinase K addition:

Add proteinase K to 60 ug/mL just prior to use in lysis (keep proteinase K frozen as long as possible). This equates to about 1.2-2 uL of proteinase K stock per 400 uL of lysis buffer.