The brews are the heart of the Catalyst reaction. This chemistry will determine the success or failure of the run. Unfortunately, this is one of the most expensive parts of the entire sequencing process. Essentially, each brew is composed of a dye primer that recognizes a sequence of the M13 vector, Taq polymerase to extend the DNA synthesis, a combi nation of nucleotides and one particular dideoxynucleotide which will ter minate the DNA elongation. Though all the primers recognize the same sequence, they have different dyes attached to them which will fluo resce at different wavelengths. Thus, for each sample, there will be four different PCR reactions. For instance, one well will have a template, all the nucleotides, plus ddATP and the primer that corresponds to the A nucleotide. Therefore, for this reaction all the DNA strands are guaran teed to start with the A primer and terminate with the A nucleotide. All the brew components are extremely temperature sensitive so keep everything on ice.
Making the 5X Buffer Solution
1-Make up a 2 M Tris (pH 8.9) solution, a 1 M ammonium sulfate solution, and a 1 M magnesium chloride solution.
2-The final concentration of the salts should be 400 mM Tris-HCl (pH 8.9), 100 mM ammonium sulfate, and 25 mM magnesium chloride.
5X Sequencing Buffer
The 5X buffer is a solution that optimizes the salt concentration for the Taq polymerase to function.
Making Taq Solution
1-Dilute the Taq with the 5X buffer and water to a final concentration of .5714 U/µl.
Diluted Taq Solution
Making the dNTP/ddNTP Mixes
1-Make the dATP/ddATP mix in a 15-ml screw cap tube to the following final concentration: 1.5 mM ddATP, 62.5 µM dATP, 250 µM dCTP, 250 µM dGTP, and 250 µM dTTP.
dATP/ddATP Mixture
2-Make the dCTP/ddCTP mixture to the following conc.: 0.75 mM ddCTP, 250 µM dATP, 62.5 µM dCTP, 250 µM dGTP, and 250 µM dTTP.
dCTP/ddCTP Mixture
3-Make the dGTP/ddGTP mix to the following final conc.: 0.125 mM ddGTP, 250 µM dATP, 250 µM dCTP, 62.5 µM dGTP, and 250 µM dTTP.
dGTP/ddGTP Mixture
4-Make the dTTP/ddTTP mix to the following conc.: 1.25 mM ddTTP, 250 µM dATP, 250 µM dCTP, 250 µM dGTP, and 62.5 µM dTTP.
dTTP/ddTTP Mixture
Making the Brews
1-Make the A brew by adding the additional reagents to the 15-ml tube; 1.38 ml 5X buffer, 1.38 ml Taq solution, and 1.38 ml of the JOE dye primer (40 pM)
A Brew
2-Make the C brew by adding the additional reagents to the 15-ml tube: 1.38 ml 5X buffer, 1.38 Taq solution, and 1.38 FAM dye primer ( 40 pM ).
C Brew
3-Make the G brew by adding the additional reagents to the 15-ml tube: 2.76 ml 5X buffer, 2.76 ml Taq solution, and 2.76 ml TAMRA dye primer ( 80 pM).
G Brew
4-Make the T brew by adding the additional reagents to the 15-ml tube: 2.76 ml 5X buffer, 2.76 ml Taq solution, and 2.76 ml ROX dye primer ( 80 pM ).
T Brew
5-Vortex the sample briefly and then spin at 1000 rpm for 1 min in the Sorvall.
6-Dispense 230 µl of each brew into en eight-well strip in the following order:
A / C / G / T / G / T / EMPTY / EMPTY
7-Store in -20°C freezer.