Short course on DNA barcoding methods
This presentation is the property of its rightful owner.
Sponsored Links
1 / 24

Short course on DNA barcoding methods November 29, 2011 PowerPoint PPT Presentation


  • 86 Views
  • Uploaded on
  • Presentation posted in: General

Short course on DNA barcoding methods November 29, 2011. DNA Extraction. Darío Lijtmaer Museo 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.

Download Presentation

Short course on DNA barcoding methods November 29, 2011

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Short course on dna barcoding methods november 29 2011

Short course on DNA barcoding methods

November 29, 2011

  • DNA Extraction

Darío Lijtmaer

MuseoArgentino de CienciasNaturales “Bernardino Rivadavia”


Short course on dna barcoding methods november 29 2011

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.


Short course on dna barcoding methods november 29 2011

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).


Short course on dna barcoding methods november 29 2011

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 obtained at the CCDB

Field work in Argentina (birds)

Tissues and vouchers deposited at MACN

Argentinian students take subsamples and are trained at the CCDB

Analysis performed together


Short course on dna barcoding methods november 29 2011

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.


Short course on dna barcoding methods november 29 2011

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.

PCR products sequenced at CCDB

Students trained

Tissues and vouchers deposited at MACN and other institutions

Field work in Argentina and neighbouring countries

Samples processed at MACN (small and medium scale)

Analysis performed together


Short course on dna barcoding methods november 29 2011

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.


Short course on dna barcoding methods november 29 2011

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


Short course on dna barcoding methods november 29 2011

Equipment: medium-sized facility

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

  • Plate scale.

Plate centrifuge

Incubator

Scale

Disposables and reagents

Pipettes

Autoclave


Short course on dna barcoding methods november 29 2011

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...

Robots

DNA extractors

Sequencing machines


Short course on dna barcoding methods november 29 2011

Overview of extraction protocols

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


Short course on dna barcoding methods november 29 2011

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.


Short course on dna barcoding methods november 29 2011

Overview of extraction protocols


Short course on dna barcoding methods november 29 2011

Overview of extraction protocols


Short course on dna barcoding methods november 29 2011

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


Short course on dna barcoding methods november 29 2011

Overview of extraction protocols: CCDB

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


Short course on dna barcoding methods november 29 2011

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.


Short course on dna barcoding methods november 29 2011

Minimizing the risk of contamination

General practices

Clean workspace and sterile tips, tubes, etc.


Short course on dna barcoding methods november 29 2011

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).


Short course on dna barcoding methods november 29 2011

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.


Short course on dna barcoding methods november 29 2011

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).


Short course on dna barcoding methods november 29 2011

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).


Short course on dna barcoding methods november 29 2011

Storage of DNA extracts

Alternatives available for long-term storage of DNA extracts

Frozen extracts

Ultracold freezer or Liquid Nitrogen

Room temperature

Dried extracts on ceramic beads or

FTA paper


Short course on dna barcoding methods november 29 2011

Questions and discussion

Thank you very much!


  • Login