cDNA synthesis
mRNA (5 μg)
primer (100p/μl) 5 μl
DW
total 25.5 μl
heat 65°C for 5 min and on ice.
5x First strand buffer 10 μl
0.1 M DTT 5 μl
10 mM dNTPs 5 μl
RNase inhibitor 2 μl
SuperScript II 2.5 μl
Total 50 μl
10 min at RT and 40°C for 1 hour and on ice
DW 232.5
5x Second strand buffer 75
10 mM dNTPs 7.5
E.coli DNA ligase (10u/μl) 2.5
Pol I (10u/μl) 10
RNase H (2u/μl) 2.5
Total volum 330
16°C 2 hours on ice
50 μl of 2.5 mM dNTPs 20
3 μl of Pfu DNA polymerase (2.5u/μl) 1.2
72°C for 30 min
phenol/Chloroform extraction and EtOH precipitation.
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