Components | Working conc. | Final conc. | 1 reaction (µl) | |
5xQ5 Reaction Buffer | 5X | 1X | 5 | |
MiFish_F | 10 µM | 0,3 µM | 0,75 | |
MiFish_R | 10 µM | 0,3 µM | 0,75 | |
dNTPs | 2 mM | 0,2 mM | 2,5 | |
Q5 HF DNA polymerase | 2 U/µl | 0.02 U/µl | 0,25 | |
Template DNA | 5 | |||
Nuclease-Free water | 10,75 | |||
Total | 25 |
A | B | C | |
Step | Temp | Time | |
Initial denaturation | 98 C | 30 sec | |
98 C | 20 sec | ||
30 cycles | 60 C | 30 sec | |
72 C | 1 min | ||
Final extension | 72 C | 7 min | |
Hold | 4 C |
Components | Working conc. | Final conc. | 1 reaction (20 μl) | |
5xQ5 Reaction Buffer | 5X | 1X | 4 | |
Forward index (i5, illu-N501-N508) | 5μM | 0.25 μM | 1 | |
Reverse index (i7, illu-N701-N712) | 5μM | 0.25 μM | 1 | |
dNTPs | 2mM | 200 μM | 2 | |
Q5 HF DNA polymerase | 2 U/μl | 0.02 U/μl | 0.2 | |
Template from 1st PCR | 2 | |||
Nuclease-Free water | 9.8 | |||
∑ | 20 |
STEP | TEMP. | TIME | |
Initial Denaturation | 98 C | 30 sec | |
98 C | 10 sec | ||
15 cycles | 66 C | 30 sec | |
72 C | 30 sec | ||
Final Extension | 72 C | 2 min | |
Hold | 6 C | ∞ |