1- 5 ml minilysate (from boiling method, 1/10 vol)
Add 15 µl Mix 1.
Incubate at 37o C for 30 min.
2- Add 10 µl (NH4)OAc (7.5 M) + 60 µl EtOH, or 12 µl 0.5 M (NH4)OAc (pH 4.5) + 75 µl EtOH.
Incubate 10 min in dry ice/EtOH bath.
Spin 15 min in Microfuge in cold room.
Wash pellet 2X with 70% EtOH.
Dry pellet in SpinVac.
3- Add 11 µl Mix 2.
Incubate at 37o C for 15 min.
Leave at room temperature for 10-15 min.
4- Add 4 µl Mix 3.
Incubate 5 min at room temp.
Place on ice.
5-Add 2.5 µl dd/dNTPs (dA, dC, dG, dT) per well of microtiter plate.
Add 3.5 µl of the mixture from Step 4 to each well.
Incubate for 10 min at 37o C.
Place on ice.
Stop the reactions with 4 µl Sanger's Stop Solution.
Prior to loading gel (6 to 8% PAGE, 8M urea), denature at 95o C for 3 min.
Mix 1: 5 µl primer (30 ng/ml), 4 µl 1 N NaOH, 6 ml ddH2O.
Mix 2: 8 µl ddH2O, 2 µl 5X reaction buffer.
Mix 3: (for 12 reactions)
22 µl ddH2O
10 µl 0.1 M DTT
10 µl 5X sequencing buffer
2 µl labeling mix
3 µl [ a-32P]dATP
3 µl Sequenase enzyme