dario lijtmaer - dna extraction
DESCRIPTION
An overview of DNA extraction protocols from small scale labs right up to high-throughput facilities.TRANSCRIPT
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Short course on DNA barcoding methodsNovember 29, 2011
DNA Extraction
Darío LijtmaerMuseo Argentino de Ciencias Naturales “Bernardino Rivadavia”
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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.
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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
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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
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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
![Page 6: Dario Lijtmaer - DNA extraction](https://reader033.vdocuments.net/reader033/viewer/2022061220/54872c95b47959fb0c8b53f3/html5/thumbnails/6.jpg)
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
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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.
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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.
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Plate centrifuge
Autoclave
Pipettes
Equipment: medium-sized facility
Scale Disposables and reagents
- Up to 20,000 thousand barcodes produced per year.
- Plate scale.
Incubator
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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
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None of the lab protocols/procedures are necessarily different from those used for other mitochondrial markers or other projects.
Overview of extraction protocols
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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
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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
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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
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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!!
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There is a similar extraction protocol developed for plants, which is also used for fungi, mollusks and echinoderms
Overview of extraction protocols: CCDB
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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
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Clean workspace and sterile tips, tubes, etc.
Minimizing the risk of contamination
General practices
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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
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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
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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
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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.
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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
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Questions and discussion
Thank you very much!