- 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.
- 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.
- Overlay with a drop of mineral oil and incubate at 60°C for 60 minutes, followed by 95°C for 15 minutes.
- 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.
- 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)
- 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).
- 60g/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 dH2O