A | B | |
Treated dsRNA | 5 µl | |
Random primers (60 µM) | 2µl | |
dNTP (10 mM) | 1 µl | |
H2O | 6 µl | |
Total | 14 µl |
A | B | |
First Strand Buffer | 4 µl | |
RNase out or RNasin® Ribonuclease Inhibitor (40 u/µl) | 1 µl | |
Maxima H minus (Point: just use 200 unit) | 1 µl | |
Total | 20 µl |
A | B | |
25 C | 20 mins | |
55 C | 90 mins | |
85 C (Inactivation step) | 5 mins |
A | B | |
cDNA | 20 µl | |
RNase H | 1 µl | |
Total | 21 µl |
A | B | |
cDNA | 21 µl | |
Klenow 10X buffer | 2.7 µl | |
dNTP (10 mM) | 1 µl | |
Klenow DNA Polymerase I | 0.7 µl | |
E. coli DNA Ligase I | 1 µl | |
H2O | 0.6 µl | |
Total | 27 µl |
A | B | |
16 C | 150 mins | |
75 C; (Inactivation step) Point: check this part based on enzyme brand. | 10 mins |
A | B | |
double stranded-cDNA | 27 μL | |
AMPure XP | 49 μL |