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B. DNA extraction
Most commercial kits use silica-based filters but a wide variety of options are available (3). For fresh animal tissues
an economical option is alkali-based extraction which is available in a kit (4) or can be home-made (5). If using a
commercial kit, simply follow the manufacturers instructions.
C. PCR
The reagents needed for PCR can be purchased as a kit or a pre-mix. Lots of choices are available and the crucial
component is the taq polymerase (6). Depending on your sample (old, degraded DNA, or fresh) and budget,
choose the best taq you can. Another critical factor can be the choice of primers. Many primers for the standard
DNA barcode regions can be found in the BOLD primer database (7). Standard PCR conditions for animal
barcoding are:
PCR RECIPE
PCR THERMOCYCLING CONDITIONS
ddH2O
16.5l
Initial denaturation 94C (1min)
10x buffer
2.5l
5 cycles: Denaturation 94C (30s), Annealing 45C
MgCl2
1.25l
(40s), Extension 72C (1min)
dNTPs
0.125l
35 cycles: Denaturation 94C (30s), Annealing 45C
Forward primer 0.25l
(40s), Extension 72C (1min)
Reverse primer 0.25l
Final extension 72C (10min)
Taq polymerase 0.12l
D. DNA sequencing
Most researchers opt to send their PCR products for sequencing by a commercial company (e.g. Macrogen, 1st
Base). As a rough guide you should expect to pay between US$5-10 for each sequence.
1. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.
2. http://botany.si.edu/projects/dnabarcode/sample2.htm
3. http://www.labome.com/method/DNA-Extraction-and-Purification.html
4. http://www.ande.com.au/
5. http://onlinelibrary.wiley.com/doi/10.1111/j.1755-0998.2009.02630.x/pdf
6. http://www.ccdb.ca/docs/CCDB_Amplification.pdf
7. http://www.boldsystems.org/index.php/Public_Primer_PrimerSearch
IMPORTANT VOCABULARY
GenBank: http://blast.ncbi.nlm.nih.gov/Blast.cgi
http://wwwabi.snv.jussieu.fr/public/abgd/
ABGD uses an
automatic recursive
procedure to
converge on the best
patterns for the
dataset and arranges
DNA barcodes into
clusters (partitions)
accordingly. The
median number of
ABDG partitions can
be used as the basis
for MOTU (species) as
this has produced
good correspondence
with traditional
species in empirical
studies.
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