Discussing the equipment, methods/protocols, PCR product verification and shipment of PCR products

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

2. Organization of the talk
1) Equipment needed for PCR amplification.
2) Overview of PCR protocols.
3) Product verification: agarose gels.
4) Minimizing the risks of contamination.
5) Shipping and storing DNA extracts.
6) Discussion and questions.

3. Equipment: basic for a small-sized facility
- Hundreds or few thousands of barcodes produced per year.
- Tube scale.
Incubator Autoclave
Thermocycler
Pipettes Disposables and
reagents
Vortex

4. Equipment: medium-sized and high throughput facilities
- Many thousand barcodes produced per year.
- Plate scale.
Incubator Autoclave
Thermocycler
Pipettes Disposables and
reagents
Vortex

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

6. Overview of amplification 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 (e.g. small PCR volumes).
b) Certain requirements are needed to achieve the barcode data standard
(e.g. minimum length).
As a consequence innovations and development of new, more efficient
protocols/proceedures are frequent in the context of the project.

7. Overview of amplification protocols: CCDB
Animals: COI
This protocol can be used with:
• Individual tubes in small-sized
facilities.
• 96 well plates in medium-sized or
high throughput facilities.
www.barcodeoflife.org

8. Overview of amplification protocols: CCDB
Plants and fungi
This protocol can be used with:
• Individual tubes in small-sized
facilities.
• 96 well plates in medium-sized or
high throughput facilities.
www.barcodeoflife.org

9. Overview of amplification protocols: PCR mix (CCDB)
Small PCR volumes: 12.5 ml for most reactions (e.g. COI in animals and
rbcL in plants), 6.25 ml for matK (plants).
• Cost-efficient.

10. Overview of amplification protocols: PCR mix (CCDB)
Small PCR volumes: 12.5 ml for most reactions (e.g. COI in animals and
rbcL in plants), 6.25 ml for matK (plants).
• Cost-efficient.
Trehalose is used as part of the PCR mix.
• It allows freezing aliquots of the mix (useful for high throughput
facilities).
• It estabilizes the reaction.

11. Overview of amplification protocols: PCR mix (CCDB)
Small PCR volumes: 12.5 ml for most reactions (e.g. COI in animals and
rbcL in plants), 6.25 ml for matK (plants).
• Cost-efficient.
Trehalose is used as part of the PCR mix.
• It allows freezing aliquots of the mix (useful for high throughput
facilities).
• It estabilizes the reaction.
Platinum taq polymerase.
• High success and band intensity, less optimization needed.
• Hot start .
• Stable at room temperature.

12. Overview of amplification protocols: PCR mix (other tips)
BSA can be added to the PCR mix to improve PCR results. This is done at
Smithsonian LAB with invertebrate samples and in the African Centre for
DNA Barcoding with plant samples.

13. Overview of amplification protocols: PCR mix (other tips)
BSA can be added to the PCR mix to improve PCR results. This is done at
Smithsonian LAB with invertebrate samples and in the African Centre for
DNA Barcoding with plant samples.
DMSO can also be added to improve PCR results with difficult samples.

14. Overview of amplification protocols: primers
Primer choice is a key aspect of PCR success and probably the only aspect
of amplification that is taxon-dependent.

15. Overview of amplification protocols: primers
Primer choice is a key aspect of PCR success and probably the only aspect
of amplification that is taxon-dependent.
Ideal situation: universal primers.
Real world: various sets of primers are to be used (and sometimes
combined) depending on the taxonomic group. There is also more than
one option for each group.

16. Overview of amplification protocols: primers
Primer choice is a key aspect of PCR success and probably the only aspect
of amplification that is taxon-dependent.
Ideal situation: universal primers.
Real world: various sets of primers are to be used (and sometimes
combined) depending on the taxonomic group. There is also more than
one option for each group.

17. Overview of amplification protocols: primers
Primer choice is a key aspect of PCR success and probably the only aspect
of amplification that is taxon-dependent.
Ideal situation: universal primers.
Real world: various sets of primers are to be used (and sometimes
combined) depending on the taxonomic group. There is also more than
one option for each group.
We included as part of the complementary materials:
the list of primers that are used at the CCDB with each taxonomic
group, the sequence of those primers and the thermocycling program that
is used with each primer.
the list of primers and the thermocycling program used at the
Smithsonian LAB.

18. Overview of amplification protocols
PCR protocols also depend on the quality of the samples used.

19. Overview of amplification protocols
PCR protocols also depend on the quality of the samples used.
For example, samples with potentially degraded DNA, such as relatively
old museum samples, require special primers designed to amplify
shorter, overlapping fragments (e.g. 200 bp long).

20. Overview of amplification protocols
PCR protocols also depend on the quality of the samples used.
For example, samples with potentially degraded DNA, such as relatively
old museum samples, require special primers designed to amplify
shorter, overlapping fragments (e.g. 200 bp long).

21. Product verification: agarose gels
PCR results are usually visualized in agarose gels.
Depending on the scale (and funding) home made gels or pre-cast gels are
used.

22. Product verification: agarose gels
PCR results are usually visualized in agarose gels.
Depending on the scale (and funding) home made gels or pre-cast gels are
used.
For plate-scales (medium scale or high-throughput) usually a threshold is
established (e.g. 75/95 bands at CCDB) and when a plate results are above
the threshold the entire plate is sequenced .

23. Mailing PCR products
If sequencing is not done on-site, PCR products are transferred to a plate
containing trehalose and dried before mailing them to a high throughput
facility.

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

25. Minimizing the risk of contamination
General practices
Clean workspace and sterile tips, tubes, etc.
Three sets of pipettes: one for extraction, one for preparing PCR and
one for PCR products (for example for gel loading).

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

27. Q&A and discussion
Thank you very much!