30 Microdissection of Chromosomes and Reverse FISH
397
Labeling mixture
Conc. stock
Final conc.
(calculated for 4 libraries)
H 2 O
Incubation mix
lOx
Ix
dNTPs
2.5 mM each
200 11M
Texas Red-dUTP
1 mM
40 11M
Primer (DOP)
40 IlM
2.0 IlM
MgCI2
25 mM
2.5 mM
Taq DNA polymerase
5 UIIlI
0.03 utili
Final volume
Volume required
53.04 III
8.50 III
6.80 III
3.40 III
4.25 III
8.50 III
0.51 III
85 III
Profile of the labeling PCR
Block temperature Time
Comments
Step 1
95°C
1 min
Denaturation
Step 2
56°C
1 min
Annealing
Step 3
noc
2 min
Extension
Repeat steps 1-320 times
Step 4
noc
5 min
Final extension
Purification
In order to separate the labeled DNA fragments from an excess of primers
and labeled nucleotides, the DNA is precipitated.
1. Add:
- 100 Ilg E. coli tRNA
- 1/10 vol 3 M sodium acetate pH 5.6
- 2 vol 100% ethanol (-20°C)
2. Mix and incubate at -20°C, 30 min
3. Cool down the centrifuge to 4°C.
4. Centrifuge at 25,000 g, 15 min, 4°C.
5. Remove the supernatant and let dry.