dario lijtmaer - dna extraction

Short course on DNA barcoding methodsNovember 29, 2011

DNA Extraction

Darío LijtmaerMuseo Argentino de Ciencias Naturales “Bernardino Rivadavia”

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.

a) Send/take tissue sub-samples for processing at a high throughput facility (different types of collaborations are possible).

Alternatives available to a person/lab interested in DNA barcoding

Both groups can be involved in the whole study...

Field work in Argentina

(birds)

Tissues and vouchers

deposited at MACN

Barcodes obtained at the

CCDBArgentinian

students take subsamples

and are trained at the

CCDB

a) Send/take tissue sub-samples for processing at a high throughput facility (different types of collaborations are possible).

Alternatives available to a person/lab interested in DNA barcoding

Analysis performed together

b) Perform the first laboratory steps of the barcoding process (extraction and amplification) in your lab and send the PCR products for sequencing.

a) Send/take tissue sub-samples for processing at a high throughput facility (different types of collaborations are possible).

Alternatives available to a person/lab interested in DNA barcoding

b) Perform the first laboratory steps of the barcoding process (extraction and amplification) in your lab and send the PCR products for sequencing.

a) Send/take tissue sub-samples for processing at a high throughput facility (different types of collaborations are possible).

Alternatives available to a person/lab interested in DNA barcoding

Field work in Argentina and neighbouring

countries

Tissues and vouchers

deposited at MACN and other

institutions Samples

processed at MACN (small and

medium scale)

PCR products sequenced at CCDB

Analysis performed together

Students trained

b) Perform the first laboratory steps of the barcoding process (extraction and amplification) in your lab and send the PCR products for sequencing.

a) Send/take tissue sub-samples for processing at a high throughput facility (different types of collaborations are possible).

Alternatives available to a person/lab interested in DNA barcoding

c) Perform the entire process in your lab.

CentrifugeVortex

Autoclave

Pipettes

Water bath or incubator

Equipment: basic for a small-sized facility

Scale Disposables and reagents

- Hundreds or few thousands of barcodes produced per year.

- Tube scale.

Plate centrifuge

Autoclave

Pipettes

Equipment: medium-sized facility

Scale Disposables and reagents

- Up to 20,000 thousand barcodes produced per year.

- Plate scale.

Incubator

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

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

Overview of extraction protocols

However...

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

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.

Overview of extraction protocols

Methods Approximate cost(per sample, USD) DNA quality

Chelex - Ethanol and Salt 0.35 poor

Commercial kits (Genelute Mammalian kit) 2.00 high

Overview of extraction protocols

Methods Approximate cost(per sample, USD) DNA quality

Chelex - Ethanol and Salt 0.35 poor

Commercial kits (Genelute Mammalian kit) 2.00 high

“Home made” glass fiber extraction 0.50 high

Overview of extraction protocols

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.

www.barcodeoflife.org

Overview of extraction protocols: CCDB

It can be used for vertebrates and most invertebrates.

Contrary to intuition, using bigger sample fragments is not better!!

There is a similar extraction protocol developed for plants, which is also used for fungi, mollusks and echinoderms

Overview of extraction protocols: CCDB

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.

Overview of extraction protocols

Clean workspace and sterile tips, tubes, etc.

Minimizing the risk of contamination

General practices

Clean tweezers between samples (ELIMINase in vertebrates, ethanol or burning in invertebrates).

Minimizing the risk of contamination

Clean workspace and sterile tips, tubes, etc.

General practices

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.

Minimizing the risk of contamination

Clean tweezers between samples (ELIMINase in vertebrates, ethanol or burning in invertebrates).

Clean workspace and sterile tips, tubes, etc.

General practices

Three sets of pipettes: one for extraction, one for preparing PCR and one for PCR products (for example for gel loading).

Minimizing the risk of contamination

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.

Clean tweezers between samples (ELIMINase in vertebrates, ethanol or burning in invertebrates).

Clean workspace and sterile tips, tubes, etc.

General practices

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).

Minimizing the risk of contamination

General practices

Three sets of pipettes: one for extraction, one for preparing PCR and one for PCR products (for example for gel loading).

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.

Clean tweezers between samples (ELIMINase in vertebrates, ethanol or burning in invertebrates).

Clean workspace and sterile tips, tubes, etc.

Frozen extracts

Ultracold freezer or Liquid Nitrogen

Room temperature

Dried extracts on ceramic beads or

FTA paper

Alternatives available for long-term storage of DNA extracts

Storage of DNA extracts

Questions and discussion

Thank you very much!