An overview of DNA extraction protocols from small scale labs right up to high-throughput facilities.

1. Short course on DNA barcoding methods
November 29, 2011
DNA Extraction
Darío Lijtmaer
Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”

2. Organization of the talk
1) Alternatives available to a person/lab interested in DNA barcoding.
2) Equipment needed for DNA extraction.
3) Overview of extraction protocols.
4) Minimizing the risks of contamination.
5) Storing of DNA extracts.
6) Discussion and questions.

3. Alternatives available to a person/lab interested in DNA barcoding
a) Send/take tissue sub-samples for processing at a high throughput
facility (different types of collaborations are possible).

4. Alternatives available to a person/lab interested in DNA barcoding
a) Send/take tissue sub-samples for processing at a high throughput
facility (different types of collaborations are possible).
Both groups can be involved in the whole study...
Barcodes
Field work in Tissues and Argentinian obtained at the
Argentina vouchers students take CCDB
(birds) deposited at subsamples
MACN and are
trained at the
CCDB
Analysis performed
together

5. Alternatives available to a person/lab interested in DNA barcoding
a) Send/take tissue sub-samples for processing at a high throughput
facility (different types of collaborations are possible).
b) Perform the first laboratory steps of the barcoding process (extraction
and amplification) in your lab and send the PCR products for sequencing.

6. Alternatives available to a person/lab interested in DNA barcoding
a) Send/take tissue sub-samples for processing at a high throughput
facility (different types of collaborations are possible).
b) Perform the first laboratory steps of the barcoding process (extraction
and amplification) in your lab and send the PCR products for sequencing.
Students PCR products
trained sequenced at CCDB
Tissues and
Field work in vouchers
Argentina and deposited at
neighbouring MACN and other Samples
countries institutions processed at
MACN (small and Analysis performed
medium scale) together

7. Alternatives available to a person/lab interested in DNA barcoding
a) Send/take tissue sub-samples for processing at a high throughput
facility (different types of collaborations are possible).
b) Perform the first laboratory steps of the barcoding process (extraction
and amplification) in your lab and send the PCR products for sequencing.
c) Perform the entire process in your lab.

8. Equipment: basic for a small-sized facility
- Hundreds or few thousands of barcodes produced per year.
- Tube scale.
Water bath or incubator
Centrifuge
Vortex
Pipettes
Scale Disposables and
reagents
Autoclave

9. Equipment: medium-sized facility
- Up to 20,000 thousand barcodes produced per year.
- Plate scale.
Incubator Plate centrifuge
Pipettes
Scale Disposables and
reagents
Autoclave

10. Equipment: high-throughput facility
- Up to hundreds of thousands of barcodes produced per year.
- Plate scale, robotic protocols.
- All pieces of equipment mentioned above plus...
DNA extractors Robots
Sequencing machines

11. Overview of extraction protocols
None of the lab protocols/procedures are necessarily different from those
used for other mitochondrial markers or other projects.

12. Overview of extraction protocols
None of the lab protocols/procedures are necessarily different from those
used for other mitochondrial markers or other projects.
However...
a) Due to the scale of the project efforts are made to reduce the cost of
the molecular steps of the pipeline.
b) Certain requirements are needed to achieve the barcode data standard.
As a consequence innovations and development of new, more efficient
protocols/proceedures are frequent in the context of the project.

13. Overview of extraction protocols
Approximate cost
Methods DNA quality
(per sample, USD)
Chelex - Ethanol and Salt 0.35 poor
Commercial kits (Genelute Mammalian kit) 2.00 high

14. Overview of extraction protocols
Approximate cost
Methods DNA quality
(per sample, USD)
Chelex - Ethanol and Salt 0.35 poor
Commercial kits (Genelute Mammalian kit) 2.00 high
“Home made” glass fiber extraction 0.50 high

15. Overview of extraction protocols: CCDB
Versions of the protocol:
• Manual with individual tubes in
small-sized facilities.
• Manual with 96 well plates in
medium-sized facilities.
• Robotic with 96 well plates in
high-throughput facilities.
It can be used for vertebrates and
most invertebrates.
Contrary to intuition, using bigger
sample fragments is not better!!
www.barcodeoflife.org

16. Overview of extraction protocols: CCDB
There is a similar extraction protocol developed for plants, which is also
used for fungi, mollusks and echinoderms

17. Overview of extraction protocols
In the case of very small invertebrates, in which the whole specimen has
to be used for DNA extraction, one option is to use a protocol that allows
the recovery of the exoskeleton as a vouchers.

18. Minimizing the risk of contamination
General practices
Clean workspace and sterile tips, tubes, etc.

19. Minimizing the risk of contamination
General practices
Clean workspace and sterile tips, tubes, etc.
Clean tweezers between samples (ELIMINase in vertebrates, ethanol or
burning in invertebrates).

20. Minimizing the risk of contamination
General practices
Clean workspace and sterile tips, tubes, etc.
Clean tweezers between samples (ELIMINase in vertebrates, ethanol or
burning in invertebrates).
If working with plates, cover all the rows of wells with caps except the
one you are working on and avoid transferring tissue above the plate.

21. Minimizing the risk of contamination
General practices
Clean workspace and sterile tips, tubes, etc.
Clean tweezers between samples (ELIMINase in vertebrates, ethanol or
burning in invertebrates).
If working with plates, cover all the rows of wells with caps except the
one you are working on and avoid transferring tissue above the plate.
Three sets of pipettes: one for extraction, one for preparing PCR and
one for PCR products (for example for gel loading).

22. Minimizing the risk of contamination
General practices
Clean workspace and sterile tips, tubes, etc.
Clean tweezers between samples (ELIMINase in vertebrates, ethanol or
burning in invertebrates).
If working with plates, cover all the rows of wells with caps except the
one you are working on and avoid transferring tissue above the plate.
Three sets of pipettes: one for extraction, one for preparing PCR and
one for PCR products (for example for gel loading).
If working with difficult samples, such as degraded DNA...
Special laboratory design (for example two separate doors that are
opened in sequence, presence of UV light).
Be extra-careful (for example, change gloves more often).

23. Storage of DNA extracts
Alternatives available for long-term storage of DNA extracts
Frozen extracts Room temperature
Ultracold freezer or Dried extracts on
Liquid Nitrogen ceramic beads or
FTA paper

24. Questions and discussion
Thank you very much!