PCR from Worm Lysate

Single worm PCR: To each 5ul worm lysate add: This lysate can also be split, 2.5ul per PCR reaction.
 * Pick an individual worm into 5ul 1X PCR buffer + 200ug/ml Proteinase K
 * Freeze at -20° or -80° for at least 30 minutes
 * Run lysis protocol on PCR machine: 1 hour 65° 15 minutes 95°
 * HOLD at 15°
 * 2ul 10X PCR buffer
 * 2.5ul 2mM dNTPs
 * .25ul TAQ
 * 1.5ul 10mM primers
 * dH20 to 25 ul

Run on PCR program: The above is 35X PCR on population of worms: Use same PCR program as above. Tm= (# of Gs and Cs in primer)*4+ (# of As and Ts in primer)*2
 * 20 sec 95°
 * 30 sec 95° (denaturing step)
 * 30 sec Tm-5° (annealing step)
 * 72° 1 min/kb of product (elongation step)
 * 72° 10 min
 * HOLD at 15°
 * Pick 5-10 L4 or adult worms into 15ul 1X PCR buffer + 200ug/ml Proteinase K
 * Freeze at -20° or -80° for at least 30 minutes.
 * Run lysis protocol on PCR machine:
 * 1 hour 65°
 * 15 minutes 95°
 * HOLD at 15°
 * Set up PCR reactions:
 * 2.5ul 10X PCR buffer
 * 2.5ul 2mM dNTPs
 * .25ul TAQ
 * 1.5ul 10mM primers
 * 1ul worm lysate
 * dH20 to 25 ul

Ideal Tm of a primer = 60°

10X PCR Buffer
 * 100mM Tris-HCl, pH 8.3
 * 500mM KCl
 * 15mM MgCl2

 Troubleshooting 

if your PCRs are not working, test several things:
 * 1) test your lysates against primers that work. the issue may be your lysate or your primers and this expmt allows you to test the lysate with primers that have worked.
 * 2) amount of template: although 1ul of worm lysate made from a population of worms usually works, you can also test 2.5ul and 5ul to see if that works better.
 * 3) altering the Mg concentration can affect your PCR. redo your PCR with a range of Mg concentrations from 1-2mM (.25mM increments).
 * 4) try a touchdown PCR:
 * 5) * 94° 30 sec
 * 6) * 10 cycles:
 * 7) ** 94° 30 sec 1o degree above the melting temperature of your primers 10 cycles and subtract 0.5° with every cycle (total of 5°; typically 61-56°) 30 sec
 * 8) ** 72° 1 min (the standard is 30 sec – 1 min/ kb of PCR product)
 * 9) * 25 cycles:
 * 10) ** 98° 30 sec 4° below the melting temperature of your primers 30 sec 25 cycles
 * 11) ** 72° 1 min (the standard is 30 sec – 1 min/ kb of PCR product)
 * 12) * 72° 10 min
 * 13) * Hold at 15°
 * 14) some primers just don’t work, especially with such a dirty lysate. order some new ones and try them.