Bio-Rad Fish DNA Barcoding Kit - Manual (Page 4)

2 Fish DNA Barcoding Quick Guide
Extracting DNA from fish samples
1. Add 200 µl of Resuspension to your two
microcentrifuge tubes containing minced fish
and flick the tubes several times to ensure
full submersion of the fish sample in the
resuspension solution.
2. Add 250 µl of Lysis to each tube and mix gently
by inverting tubes 10 times to mix contents. Do
not vortex! Vortexing may shear genomic DNA,
which can inhibit PCR amplification.
3. Incubate samples at 55°C for 10 min. The
samples do not need to be shaken during
incubation.
4. Add 250 µl of Neutralization to each
microcentrifuge tube and mix gently by inverting
tubes 10 times to mix contents (do not vortex). A
visible cloudy precipitate may form.
5. Centrifuge the tubes for 5 min at top speed
(12,000–14,000 x g) in the microcentrifuge. A
compact pellet will form along the side of the
tube. The supernatant contains the DNA.
If there are a lot of particulates remaining in the
supernatant after centrifugation, centrifuge
the tubes for 5 additional min.
6. Snap (do not twist!) the bottoms off of the spin
columns and insert each column into a capless
2 ml microcentrifuge tube.
7. Label one spin column 1 for Fish 1 and a second
spin column 2 for Fish 2. Also label the columns
with your initials.
Invert
gently, 10x
55°C Water bath
10 min
Resuspension
200 µl
Flick
1 2
Lysis
250 µl
1 2
Invert
gently, 10x
Neutralization
250 µl
1 2
1 2
12,000–14,000 x g
5 min