30 Microdissection of Chromosomes and Reverse FISH
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2. Prepare reaction Mix C. Approximately 2 f.!l are needed for each reaction. If 4 tubes are to be amplified prepare 10 f.!l.
Reaction mix C
Cone. stock
Sequenase dilution buffer
Sequenase Version 2.0
8x
Final cone.
Ix
Volume required
8.75 III
1.25 III
3. Insert tubes into the thermocycler and start the program (see below).
The first step is a thorough denaturation of the DNA, which at the same
time inactivates the Proteinase K.
4. During the initial 8 cycles 0.24 III (about 0.3-0.4 units Sequenase) of
reaction mix C is added at each annealing step (step 2). This is necessary, because the Sequenase is not thermostable and becomes inactivated during each denaturation step.
5. Prepare reaction mix D.
6. Add 50 f.!l of reaction mix D to each tube during step 5 (see below). If
necessary increase the amount of mineral oil in the reaction tube.
Reaction mix D
Cone. stock
Final cone.
Volume required
H 2 O
142.48 III
Incubation mix
lOx
Ix
21.0 III
dNTPs
2.5 mM each
220 llM
18.48 III
Primer (DOP)
40 llM
l.lIlM
5.78 III
MgCl2
25mM
2.5 mM
21.0 III
Taq DNA polymerase
5 Uilli
0.03 Ullll
1.26 III
Final volume
210.0 III