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DNA ExtractionCCDB Protocols for High-Throughput Barcoding

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Our Presenter

Natalia Ivanova received her Ph.D. in Molecular Biology from Lomonosov Moscow State University in 1998. Most of her Ph.D. data on the molecular systematics of lichens was gathered at the NMNH, Smithsonian Institution. In 2004 Natalia joined the Hebert laboratory and contributed to the development of cost-effective high throughput barcoding protocols and the integration of robotics into the analytical chain. She currently oversees automation, core lab troubleshooting, the R&D lab, and forensic barcoding at the Canadian Center for DNA Barcoding.

Natalia IvanovaLead DNA Scientist, CCDB



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DNA Extaction



Our Agenda

• Sampling standardization for high-throughput

• Classification of DNA extraction protocols

• CCDB DNA extraction protocols

• Mix & match Glass Fiber DNA extraction protocol

• Voucher recovery

• Manual vs. automated extraction

• Shipment of DNA and PCR products

• Questions and answers



direct sampling into microplates

Require subsampling Lab-ready!

2004 2008

tube racks 12x8

High Throughput Sampling Protocols: CCDB Example



High Throughput Sampling Protocols: Plants

1. Silica gel preserved material2. When sampling into a specimen

array, tissue amount is important 3. A-B – correct4. C-D – incorrect

=

+ 3 4

2

1



High Throughput Sampling Protocols: FTA cards

FTA FTA Elute

Pathogen inactivation Yes Yes

Room temperature storage and shipment Yes Yes

DNA elution No (only by pH change) Yes

Contaminants Washed away Remain on a filter

Extraction protocol 30 min – 1.5 hours 5 min

Additional reagents Yes No (only water)



Overview of DNA ExtractionTissue +

Lysis Buffer + ProK

DNA and contaminants released into

solutionAdjust binding

conditionsBind DNA to membrane

1st WashDry the

membrane and elute DNA

Add phenol/chloroform, centrifuge

Transfer top phase, adjust precipitation conditions

Centrifuge to pellet DNA at the bottom

Remove supernatant

Wash DNA with 70%

EtOH

Remove supernatant

Dissolve DNA

56 C

2nd Wash



Extraction Protocols Not Used At the CCDB

SPRI – magnetic beads

Image from Seradyn web-site

OpenWetWare imageDNA

Contaminants

Size exclusion

Phenol/Chloroform– ethanol precipitation



Extraction Protocols Used By the CCDB

Bind-wash-elute protocols (membrane based)

Crude lysis (Chelex, alkaline lysis) FTA cards



CCDB Protocols:Chelex and Alkaline Lysis

• Simple, quick, inexpensive

• Suitable for very small organisms

• Do not produce stable DNA extracts

• Tissue amount is critical

• HotSHOT protocol for rotifers, tardigrades and small crustaceans



• Lysis – 25-50 µL of 25 mM NaOH, 0.2 mM EDTA, pH 8.0• Incubate for 30 min at 95 C• Neutralization – equal volume of 40 mM Tris-HCl , pH 5.0• Mix, use 1-2 µL in PCR• Store DNA extract for up to 3 years at -20 C

CCDB Protocols:HotShot Alkaline Lysis



1. Apply sample; dry

2. Punch a disc and place in a tube

3. Rinse punch in water for 5 sec

4. Remove water

5. Use disk in PCR OR add water, heat at 95 C for 15 min; pulse vortex; centrifuge

6. Use eluted DNA in PCR

FTA Elute 96 Well Card – Whatman WB12 9231; Fisher FSSP9777052

CCDB Protocols: FTA Elute Cards



CCDB Protocols:Glass Fiber DNA Extraction

EcoRI digestion of plant DNA



Lysis Buffers

• Invertebrate

• Vertebrate

• CTAB

• Algal Buffer (Saunders lab)



Binding Conditions

Plant Binding Buffer (5M GuSCN buffer)

Binding Mix (3M GuSCN buffer, 50% ethanol)



Binding Media

1 µm Glass fiber – PALL #5051

3 µm Glass fiber over 0.2 µm Bioinert – PALL #5053



Invertebrate Extraction • Tissue source – legs or whole specimens

• Invertebrate lysis buffer + Proteinase K

• Incubation overnight at 56 C

• Binding with BM buffer, wash with PWB and WB buffers

• 3 µm GF over 0.2 µm Bioinert membrane

• Elution volume – 30-40 µl



Vertebrate Extraction • Tissue source – ideally muscle

• Vertebrate lysis buffer + Proteinase K


• Binding with BM buffer, wash with PWB and WB buffers

• 1 µm GF membrane




Plant and Fungal Extraction • Tissue source – silica dried tissue, herbarium

samples, seeds

• Grinding – TissueLyser

• CTAB lysis for 1.5 hour at 65 C

• Binding with 5M GuSCN buffer, wash with BM and WB buffers

• 1 µm GF membrane




Echinoderm and Mollusk Extraction

• Tissue source – ethanol fixed soft tissue

• CTAB + Proteinase K lysis



• 3 µm GF over 0.2 µm Bioinert membrane




Algal DNA Extraction



Algal Extraction – Saunders Lab/CCDB • Tissue source – silica dried tissue, herbarium• Acetone extraction to remove polyphenols• Grinding – TissueLyser

• Algal buffer + Tween 20 + Proteinase K, incubate 1 hour at room temperature

• Incubate on ice, centrifuge• Transfer aliquot of lysate


• 1 µm GF membrane• Elution volume – 50-60 µl



Voucher Recovery Protocol

Collembola after 12H incubation



Voucher Recovery Protocol

1. Incubate overnight in a lysis buffer

2. Apply lysate to 350 µl PALL plate with 0.45 µm GHP membrane sitting on top of collection plate with PALL collar

3. Centrifuge at 3000 g for 2 min

4. Use clarified lysate for extraction

5. Add 95% EtOH to lysis and PALL plates

6. Collect vouchers under microscope



Manual Extraction

• Scalable for 4-6 plates/day

• Centrifugation at 5000 g

• Better for old material

• Less chances of contamination



Automated & Semi-Automated Extraction

• 20-40 plates/day

• Vacuum manifold

• Centrifugation to remove WB

• Elution in centrifuge

• Possible integration of robotic centrifuge

• Less errors



PCR Optimization

• Pre-made frozen plates with trehalose• Low dNTPs and primers concentration• Platinum or KAPA Ab Taq polymerase• No PCR cleanup!



DNA and PCR Products Shipment

• DNA & PCR products exchange between iBOL nodes• Backup system for freezers



Trehalose and Preservation

1994. Trends Ecol. Evol. 9: 230



Biomatrica – Emerging Technology



CCDB: 1 Year Storage Experiment

Mean Contiguous Read Length (bp)

480

500

520

540

560

580

600

Fridge +4 Freezer -20 Biomatrica RT Biomatrica 56 Trehalose RT Trehalose 56



DNA and PCR Products Recommendations

• Convenient shipment of DNA and PCR products at room temperature

• Backup options for -20 or -80 C archiving

• Trehalose is sufficient for shipment

• Biomatrica is a better choice for forensic, degraded or diluted samples

• More data is needed to advocate transition to dry storage at room temperature



CCDB Protocols

http://www.dnabarcoding.ca/pa/ge/research/protocols



Bibliography• deWaard JR, Ivanova NV, Hajibabaei M, Hebert PDN (2008) Assembling DNA barcodes: analytical protocols. In:

Environmental Genomics, Methods in Molecular Biology, vol. 410 (ed. Martin CC), pp. 275-283. Humana Press.• Hajibabaei M, DeWaard JR, Ivanova NV, Ratnasingham S, Dooh RT, Kirk SL, Mackie PM, Hebert PDN (2005) Critical

factors for assembling a high volume of DNA barcodes. Philosophical Transactions of the Royal Society of London B Biological Sciences 360, 1959-1967.

• Ivanova N, Kuzmina M and Fazekas A (2011) CCDB Protocols, Glass Fiber plate DNA extraction for plants, fungi, echinoderms and mollusks. Retrieved from http://www.ccdb.ca/CCDB_DOCS/CCDB_DNA_Extraction-Plants.pdf on April 8, 2011.

• Ivanova NV, DeWaard JR, Hebert PD (2007) CCDB Protocols, Glass Fiber Plate DNA Extraction. Retrieved from http://www.dnabarcoding.ca/CCDB_DOCS/CCDB_DNA_Extraction.pdf on April 7, 2011.

• Ivanova NV, deWaard JR, Hebert PDN (2006) An inexpensive, automation-friendly protocol for recovering high-quality DNA. Molecular Ecology Notes 6, 998-1002.

• Ivanova NV, Fazekas AJ, Hebert PDN (2008) Semi-automated, Membrane-Based Protocol for DNA Isolation from Plants. Plant Molecular Biology Reporter 26, 186-198.

• Ivanova NV, Grainger CM (2007) CCDB Protocols, COI amplification. Retrieved from http://www.dnabarcoding.ca/CCDB_DOCS/CCDB_Amplification.pdf on April 7, 2011.

• Kuzmina M, Ivanova N (2011) CCDB Protocols, PCR amplification for plants and fungi. Retrieved from http://www.dnabarcoding.ca/CCDB_DOCS/CCDB_Amplification-Plants.pdf on April 8, 2011.

• Porco D, Rougerie R, Deharveng L, Hebert PDN (2010) Coupling non-destructive DNA extraction and voucher retrieval for small soft-bodied Arthropods in a high-throughput context: the example of Collembola. Molecular Ecology Resources 10, 942–945.

• Saunders GW (1993) Gel purification of red algal genomic DNA: an inexpensive and rapid method for the isolation of polymerase chain reaction-friendly DNA. Journal of Phycology 29, 251-254.

• Saunders GW (2005) Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications. Philosophical Transactions of the Royal Society B-Biological Sciences 360, 1879-1888.

• Whitlock R, Hipperson H, Mannarelli M, Burke T (2008) A high-throughput protocol for extracting high-purity genomic DNA from plants and animals. Molecular Ecology Resources 8, 736-741.


http://www.ccdb.ca/CCDB_DOCS/CCDB_DNA_Extraction-Plants.pdf�

http://www.dnabarcoding.ca/CCDB_DOCS/CCDB_DNA_Extraction.pdf�

http://www.dnabarcoding.ca/CCDB_DOCS/CCDB_Amplification.pdf�

http://www.dnabarcoding.ca/CCDB_DOCS/CCDB_Amplification-Plants.pdf�


Glass Fiber Automated DNA Extraction Video

Launch 7.5 minute video: BiomekFX in Action



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DNA Extraction - Ningapi.ning.com/files/Qsv6Z0-p8iv-2UHJVuMWhwXw84JwdQp2fZXnipwQ3XikgrE...Algal DNA...

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