A | B | |
1ml of Incubation buffer composition | ||
H20 | 460 μl | |
20% Ficoll PM-400 (GE healthcare) | 300 μl | |
500mM EDTA (Life Technologies) | 40 μl | |
1M Tris pH 7.5 (Sigma) | 200 μl | |
Total | 1000 μl |
A | B | |
RT mix composition | ||
H20 | 80 μl | |
Maxima 5X RT buffer | 40 μl | |
20% Ficoll PM-400 | 40 μl | |
10mM dNTPs | 20 μl | |
100uM TSO | 5 μl | |
RNase inhibitor | 5 μl | |
Maxima H- RTase | 10 μl | |
Total | 200 μl |
A | B | |
Exonuclease mix composition | ||
10X ExoⅠbuffer | 20 μl | |
H20 | 170 μl | |
ExoⅠ | 10 μl | |
Total | 200 μl |
A | B | |
RT mix composition | ||
H20 | 80 μl | |
Maxima 5X RT buffer | 40 μl | |
20% Ficoll PM-400 | 40 μl | |
10mM dNTPs | 20 μl | |
100uM TSO | 5 μl | |
RNase inhibitor | 5 μl | |
Maxima H- RTase | 10 μl | |
Total | 200 μl |
A | B | |
Exonuclease mix composition | ||
10X ExoⅠbuffer | 20 μl | |
H20 | 170 μl | |
ExoⅠ | 10 μl | |
Total | 200 μl |
A | B | |
cDNA amplification PCR composition | ||
DW | 14.6 μl | |
beads in DW | 10 μl | |
SMART PCR Primer (100uM) | 0.4 μl | |
2X KAPA | 25 μl | |
Total | 50 μl |
A | B | |
95C 3min | ||
Denature | 98C 20 sec | |
Annealing | 65C 45 sec | |
Elongation | 72C 3 min | |
4 cycles | ||
Denature | 98C 20 sec | |
Annealing | 67C 20 sec | |
Elongation | 72C 3 min | |
9 cycles | ||
Final extension | 72C 5 min | |
Hold | 4C forever |