Sequencing Reaction Procedure  

Annealing temperatures are T = 35, 47.5, 60 (code C=1,2,3).

• Prepare 3 PCR machines.
• Alliquot each reaction mix into 3 tubes (4.5 ul each), one for each temperature (36 total).
• Overlay reaction tubes with oil to prevent evaporation.
• Place in PCR machines according to temperature.

• Begin program: raise machine to 98C for 5 min to denature KSII; then decrease temperature to (80?) and hold.
• Add 1 ul of 0.5 U/ul Taq DNA polymerase (in 1X Taq seq. buf.) to each tube, while holding at (80?). Mix by pipetting.

          Final annealing volume: 5.5 ul
          Final annealing concentrations:           total amt per tube
                  KSII        40.9 ng/ml (20.45 nM) .11 pmol, 68B molec.
                  primer      100 nM                .45 pmol, 271B molec
                  Taq buf     1 X
                  [a35S]dATP  0.818 mCi/ml          4.5 uCi
                  Sanger mix  0.545 X
                  Taq polym.  0.182 U/ul            0.5 U
  

• When ready, begin new program: cool to annealing temperature T (annealing and polymerization begin during cooling) and hold.
• Keep at T for at least 10 min. to allow ample time for annealing and polymerization to proceed. (Annealing half-life estimated to be 1.5 minutes.)
• After ~10 min. at T, add 1 ul dNTP chase to each tube. Mix by pipetting.
• When ready, begin new program: heat to (70?) C to speed polymerization, and hold at that new temperature.
• After ~10 min. at (70?), add 6 ul stop/loading dye to each tube.
• When done, turn off machines, remove reaction products, store on ice for up to 1 week.