396
ILSE CHUDOBA and GABRIELE SENGER
PCR-profile
Block temperature Time
Comments
Step 1
95°C
5 min
Initial denaturation
Step 2
30°C
2 min
Preamplification annealing
Step 3
37°C
2 min
Preamplification extension
Step 4
95°C
1 min
Preamplification denaturation
Repeat steps 2-4 8 times
Step 5
30°C
2 min
Transition
Step 6
95°C
1 min
Denaturation
Step 7
56°C
1 min
Annealing
Step 8
noc
2 min
Extension
Repeat steps 6-8 30 times
Step 9
noc
5 min
Final extension
In order to protect the DNA from nuclease degradation, EDTA is added to
a final concentration of SmM. For long term storage, the tubes are frozen
at -20°C.
Labeling of DOP-amplified DNA libraries
The DNA fragments resulting from the initial preamplification are all
tagged with the DOP-primers. Therefore, the same primers can be used
in order to amplify the DNA products and incorporate labeled nucleotides. Either Biotin- or Digoxigenin-Iabeled nucleotides as well as nudeotides directly coupled with fluorochromes can be used. By incorporation
of directly labeled nudeotides, any time consuming post-hybridization
detection steps are avoided and hence, preferred. In the following protocol
we use Texas Red labeled dUTPs but Biotin- or Digoxigenin-dUTP as well
as other directly labeled nudeotides can be incorporated. For each microdissection library 20 III oflabeling mixture is prepared. Add 2 III of the PCR
product from the first PCR.