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PCR on Worms

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PCR on Worms

(adapted from Mike Krause)

1. Harvest worms (1 plate is plenty) in 1-2 ml of water into a 1.5ml eppendorf tube.

2. Spin (1 min, 15,000 rpm), and remove excess water without disturbing worm pellet.

3. Add 200 ul of lysis buffer to worm pellet.

Lysis buffer

10mM Tris pH 8.3

50mM KCl

1.5mM MgCl2

0.45 % Tween

0.45 % NP40

100ug/ml Proteinase K

4. Incubate at 65oC for one hour with occasional shaking

5. Incubate at 95oC for 15 min (to kill proteinase K)

6. Set up PCR reaction as follows (mix reagents on ice):

DNA (digested worms) 2.0ul

10X Taq buffer 5.0ul

25mM MgCl2 3.0ul

25mM dNTPs 0.4ul

Primer 1 (50ng/ul) 4.0ul

Primer 2 (50ng/ul) 4.0ul

H20 31.1ul

Taq (Promega 5u/ul) 0.5ul

7. Cycle PCR reaction as follows

Step 1 94oC 1 min

Step 2 56oC 2 min

Step 3 72oC 5 min

Step 4 94oC 45 sec

Step 5 56oC 45 sec

Step 6 72oC 1 min 30 sec

Repeat Steps 4-6 34 times

Step 7 72oC 15 min

8. Load 15 ul on 1% agarose gel