PCR from Worms
¯ Pick one worm and place it in a 2.5ml 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 –700C for 15min. 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 600C for 60 minutes, followed by 950C for 15 minutes.
¯ Cool to 40C. Pipette 22.5ml of PCR ÒMaster MixÓ 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 940C and cycle 30 times through a program appropriate for the template DNA and the primers you are using in the reaction:
Melting step: 940C for 30 sec (standard)
Annealing step: ~50C below melting pt. of the primers and usually held for between 30 seconds and 1 min.
Extension step: 720C standard temp. and est. time based on length of template (approx. 1 min per 1 kb)
¯ Analyze 10ml of each sample on a 3.0% ÒMetaphor agarose gel, or a 6% acrylamide gel. Be sure to run lstyI or some other molecular weight marker.
Lysis Buffer- (store on the bench top w/o proteinase K)
¬ 200mg/ml proteinase K
¬ 10mM Tris-Cl, pH 8.2
¬ 50mM KCl
¬ 2.5 mM MgCl2
¬ 0.45% Tween 20
¬ 0.05% gelatin
Prepare a Master Mix containing the following amounts of each component PER REACTION. Make enough Master Mix for N+1 reactions.
¬ 2.5 ml 10X Amplification Buffer
¬ 2.5 ml 2mM dNTPs
¬ 1.5 ml 25mM MgCl2
¬ 0.12 ml 5 U/ml TAQ Polymerase (0.6 Units/reaction)
¬ 1.0 ml Primer Mix (25pMol/ml conc. of primer combination)
¬ 15.88 ml dH2O