Bio-Rad Fish DNA Barcoding Kit - Manual (Page 7)

Fish DNA Barcoding Quick Guide 5
Quick Guide
Lesson 2: PCR Amplification of DNA
1. Label four PCR tubes with your initials and the
sample name (1 for fish sample 1, 2 for fish
sample 2, (+) for the PCR positive control DNA,
(–) for the PCR negative control). Keep the tubes
on ice for the remaining steps.
2. Using a fresh aerosol filter pipet tip each time,
add 35 µl of CMM (COI master mix) reaction
mix to each PCR tube, capping each tube
immediately after the addition of liquid.
3. Using a fresh aerosol filter pipet tip for each
tube, add 5 µl of the appropriate DNA sample
directly into the CMM liquid in each PCR tube
as indicated by the labels on the tubes, and
pipet up and down to mix. Recap each tube
immediately after adding DNA.
Tube Name Master Mix DNA
1 35 µl CMM, 5 µl fish sample 1
2 35 µl CMM, 5 µl fish sample 2
(+) 35 µl CMM, 5 µl (+) sample
(–) 35 µl CMM, 5 µl (–) sample
4. When instructed, place the PCR tubes in the
thermal cycler and run the program with the
following cycling conditions:
1. 94°C – 2 min
2. 94°C – 30 sec
3. 55°C – 2 min
4. 72°C – 1 min
5. Repeat steps 2–4 35x
6. 72 °C – 10 min
7. 4°C – hold
Store tubes at 4°C after thermal cycling is
complete.
35 µl
CMM
1 2 (+) (–)
1 2 (+) (–)
1
5 µl