Bio-Rad Fish DNA Barcoding Kit User Manual

Biotechnology Explorer

Fish DNA Barcoding Kit

Quick Guide

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Catalog #166-5100EDU

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Quick Guide

Lesson 1: DNA Extraction

Preparing Fish Samples

1. Label one capped 2 ml microcentrifuge tube for
each of your fish samples (that is, “1” for fish
sample 1, “2” for fish sample 2, etc.). Also label
with your initials.

Fish 1__________________

Fish 2__________________

2. Cut a piece of fish muscle up to 100 mg in
mass, approximately the size of a pencil eraser­head, from your first fish sample. Place the piece
in a new weigh boat and slice it with a razor
blade or cutting implement until finely minced.
Transfer the sample into the appropriately
labeled microcentrifuge tube.

1

Initials Initials

1

2

3. Properly discard the razor blade or cutting
implement. If wearing gloves, change gloves

before handling the next piece of fish. If not,
wash hands thoroughly.

4. Using a new razor blade or cutting implement,
cut a piece of fish muscle up to 100 mg in mass,
approximately the size of a pencil eraser-head,
from your second fish sample. Place the piece
in a new weigh boat and slice it with a razor
blade until finely minced. Transfer the sample
into the appropriately labeled microcentrifuge
tube. Properly discard the razor blade or cutting
implement.

2

Fish DNA Barcoding Quick Guide 1

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.

200 µl

Resuspension

250 µl

Lysis

250 µl

1

1

55°C Water bath

2

2

10 min

Flick

Invert

gently, 10x

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.

Neutralization

12,000–14,000 x g

5 min

1

1 2

2

Invert

gently, 10x

2 Fish DNA Barcoding Quick Guide