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Primers

Autorads

Data

Genotypes

OSU-Barley

(2) The Taq comes with 25 mM MgCl2. Dilute this to 20 mM (this represents a 10X stock).

(3) Prepare a dNTP stock solution of 1.6 mM: dCTP, dGTP, dTTP; with 50 M dATP. This is if you are using a 33P[a]dATP. I call this my 10X dNTP mix (-A).

(4) You can use dCTP, but you need to make a (-C) dNTP mix.

(5) After everything is aliquoted into thermocycler tubes you can cap tubes and place into the thermocycler and run the following program: 94^o, 3'; 55^o ,2'; and 72^o,1.5' for one cycle, followed by 30 cycles of 94^o, 1'; 55^o, 2'; 72^o, 1.5'; and ending with an extension of 72^o, 5'.

(6) When the program has finished the run (about 3 hours) you can add 20 l of STOP (95% formamide, 20 mM EDTA, 0.05% Bromophenol Blue, 0.05% Xylene Cyanol) solution per tube. (You can freeze the reactions -20^o C for up to one week.)

(7) Denature the samples @ 94^oC for 8 minutes (create a thermocycler program for this also).

(8) Run out the samples on a 6% sequencing gel (pre-run the gel for ~30 minutes). Run the gel: 65 watts (running at higher wattage may overheat the glass plates, and cause either a crack to form or glass warping) constant power; until the Xylene Cyanol is in the center of the gel (this is just an approximation, your gel rig may differ).

(9) Dry the gel and expose to x-ray film overnight.

(10)***If the reactions did not work well check the Tm of the primers, or try using the "Touchdown" program: 94^o, 1'; 64^o, 30"; and 72^o for 1'. The annealing temp. is decreased 1^o every second cycle until the temp is 55^o. After 55^o is reached the reaction is run through 30 cycles of: 94^o, 1'; 55^o, 1' and 72^o, 1' with a final extension of 72^o, 5'.

Electrophoresis

A. You will need to make or purchase 6% acrylamide solution, sequencing mix, and 10% APS. You should try to keep a stock of 40%* (19% acrylamide:1% bis-acrylamide) in the lab.

(* 40% stock is 38 grams acrylamide, 2 grams methylene-bis-acrylamide for 100 mls of solution made with sterile dH2O.)

2) Use sterile H2O to bring to volume, filter, and refrigerate the acrylamide until needed.

3) For the APS (ammonium persulfate) make 10 mls by adding 1 gram APS to sterile H2O and bring to volume.

4) For pouring a gel, use 1 ml of 10% APS + 25 l TEMED/100 mls acrylamide (these are approximate amounts of TEMED & APS) solution.

B. Cleaning the glass plates

1) Use as non-abrasive a method as possible, I prefer using an ordinary glass cleaner (e.g. "Windex"), then rinsing with dH2O, and then rinse with 95% EtOH and wiping dry.

2) Apply siliconizing agent ("Rain-X", "Sigmacote", etc.) to the short (or dog-eared) plate. You should buff this on and then wipe down with a damp kimwipe, rinse with EtOH.

3) Store the plates cleaned and ready for the next gel by clamping together (with spacers in position) and wrapping ends of plates with plastic-wrap.