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DNA Cycle Sequencing
The
Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham
Life Science) is used.
1.
Treat PCR products with enzymes exonuclease I and Shrimp Alkaline
Phosphatase (SAP) i.e.
5
ml
PCR product
1
ml
Exonuclease I
1
ml
SAP (shrimp alkaline phosphatase)
2.
Incubate at 37oC for 15 minutes, followed by 80oC
for 15 min
3.
Label strips of eppendorfs - 4 (G, A, T and C) for each sample
4.
Prepare termination mixtures i.e. for 12 tubes:
G = 24
ml
Termination master mix + 6
ml
of ddGTP
A =
กฐ + 6
ml
of ddATP
T =
กฐ + 6
ml
of ddTTP
C =
กฐ + 6
ml
of ddCTP
5.
Aliquot 3
ml
of DNA and 17
ml
reaction mixtures, mix and dispense 5
ml
into each of the tubes labelled G, A, T, and C.
6.
Prepare the reaction mixtures
Per sample 12 samples
Reaction buffer 2.0
ml
24
ml
Primer 0.5
ml
6
ml
Water 12.5
ml
150
ml
Thermo
Sequenase 2.0
ml
24
ml
7. Add
2.5
ml
of the appropriate termination mixes into each of the tubes, mix
well and cover with oil.
8.
Carry out cycle sequencing using GeneE progam 38 for 1.5 hours (dGTP
system only)
30 cycles 95oC
for 30s, 55oC for 30s, 72oC for 60s
1 cycle 20oC
holding temp
9.
Aliquot 6ml
of product form each tube into wells of a microtitre plate, and
add 4
ml
of stop solution to terminate the reaction. Seal the microtitre
plate with a plate sealer.
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