46
KAI-OLAF NETZER
3. Place on ice for 2 min. Centrifuge at 12000 rpm in a microcentrifuge for
30 sec.
4. On ice, add the following to the reaction mixture:
10x buffer
2 !ll
dNTP mix
1 !ll
10 nmol each dNTP
DTT
2 !ll
0.211mol
RNasinTM
1 !ll
MMLV-RT
1 !ll
200 u
5. Mix gently. Collect the reaction mix by brief centrifugation.
6. Incubate at 37°C for 1 hour.
7. lncrease the temperature to 65°C for 5 min. Place on ice for 2 min. Collect
the reaction by brief centrifugation.
8. Remove 5 111 of the first strand synthesis reaction to a new 0.5 ml tube on
ice. Store the remainder at -20°C.
Note: The newly synthesized cDNA may be directly amplified by PCR using
specific amplification primers. If maximum sensitivity is required, use 20 111
of the firststrand reaction as template for a 100 111 PCR amplification.
9. Add
10x buffer
4.5 !ll
The same buffer as above
maybe used.
PCR primer 1
1 !ll
20 pmol
PCR primer 2
1 !ll
20 pmol
dNTP mix
6 !ll
10 nmol each dNTP
Taq polymerase
0.5 !ll
2.5 u
Water
32 !ll
10. Overlay the reaction with 50 111 mineral oil.
11. Perform 20 to 40 rounds of PCR with the appropriate temperature/time
prof:lle.
12. Analyze the amplified cDNA product on a 1.5-2.0% agarose gel.