Worm PCR

 

Worm PCR

1. Pick one worm and place it in a 2.5 l drop of lysis buffer in the cap of a PCR tube. Close and centrifuge briefly to move to the bottom of the tube.
2. Freeze the tubes at -70°C for 15 min. The idea is to do a freeze-crack to help liberate the DNA. Check that the solution actually freezes.
3. Overlay with a drop of mineral oil and incubate at 60°C for 60 minutes, followed by 95°C for 15 minutes.
4. Cool to 4°C. Pipette 22.5 l of PCR master mix onto the top of the mineral oil overlay. Set sample in ice bucket until all samples have been prepared.
5. Microfuge briefly to move the master mix through the mineral oil overlay. Rapidly heat the samples to 94°C and cycle 30 times through a program appropriate for the template DNA and the primers you are using in the reaction:
   • Melting step : 94°C for 30 sec (standard)
   • Annealing step : ~5°C below melting pt. of the primers and usually held for between 30 seconds and 1 min.
   • Extension step : 72°C standard temp. and est. time based on length of template (approx. 1 min per 1 kb)
6. Analyze 10 ml of each sample on a 3.0% Metaphor agarose gel, or a 6% acrylamide gel. Be sure to run Sty I or some other molecular weight marker.

Lysis buffer (store on the benchtop without proteinase K).

• 60 g/ml proteinase K
• 10mM Tris-Cl, pH 8.2
• 50mM KCl
• 2.5mM MgCl₂
• 0.45% Tween-20
• 0.05% gelatin

Master mix . Prepare a master mix containing the following amounts of each component per reaction. Make enough master mix for (n+1) reactions.

• 2.5 l 10X amplification buffer
• 2.5 l 2mM dNTPs
• 1.5 l 25mM MgCl₂
• 0.12 l 5U/ l Taq polymerase (0.6 Units/reaction)
• 1.25 l primer mix (20pMol/ l conc. of primer combination)
• 14.63 l dH₂ O