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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
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