DNA Extraction, Preservation, and Amplification

Chapter 14 DNA Extraction, Preservation, and Amplification Thomas Knebelsberger and Isabella Stöger Abstract The effectiveness of DNA barcoding as a routine practice in biodiversity research is strongly dependent on the quality of the source material, DNA extraction method, and selection of adequate primers in combination with optimized polymerase chain reaction (PCR) conditions. For the isolation of nucleic acids, silicagel membrane methods are to be favored because they are easy to handle, applicable for high sample throughput, relatively inexpensive, and provide high DNA quality, quantity, and purity which are prerequisites for successful PCR amplification and long-term storage of nucleic acids in biorepositories, such as DNA banks. In this section, standard protocols and workflow schemes for sample preparation, DNA isolation, DNA storage, PCR amplification, PCR product quality control, and PCR product cleanup are proposed and described in detail. A PCR troubleshooting and primer design section may help to solve problems that hinder successful amplification of the desired barcoding gene region. Key words: DNA barcoding, DNA extraction, DNA preservation, PCR amplification, Agarose gel electrophoresis, PCR cleanup

1. Introduction The extraction of genomic DNA requires careful sample preparation, followed by tissue lysis and isolation of the nucleic acids. The lysis of the tissue samples is performed by applying enzymatic digestion, commonly with Proteinase K, which degrades proteins and rapidly inactivates nucleases that might otherwise degrade DNA during isolation and purification. After digestion, nucleic acids are separated from all other remaining cellular components. The condition of the biological source material plays a pivotal role for the quality, quantity, and purity of the extracted DNA. Therefore, appropriate tissue or sample storage after collecting the biological source material in the field is required. Besides the use of recently collected and preserved organisms, museum

W. John Kress and David L. Erickson (eds.), DNA Barcodes: Methods and Protocols, Methods in Molecular Biology, vol. 858, DOI 10.1007/978-1-61779-591-6_14, © Springer Science+Business Media, LLC 2012

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specimens represent a convenient source for species-wide samplings. But nucleic acids might be highly degraded due to either extensive exposure to killing agents, like acetate, ethyl alcohol, or cyanide (1–3), or sample storage under inappropriate conditions; fixatives, like formaldehyde or other aldehyde mixtures often used in museums to preserve biological material, degrade DNA, which makes the extraction of utilizable nucleic acids quite challenging (4–8). For routine DNA extraction, the methods based on silica-gel membrane technology have proven to yield DNA of high quantity and quality (9). In comparison to other extraction technologies, these methods are easy to handle, relatively inexpensive, and allow high sample throughput (96-well format). After enzymatic digestion of the samples, nucleic acids are adsorbed to a silica-gel membrane in the presence of highly concentrated chaotropic salts. Fragment lengths up to 20 kb can be recovered in high purity usable for downstream applications as well as for long-term storage. Other DNA extraction technologies, like salting out precipitation or anion exchange methods, yield DNA fragment lengths up to 150 kb but are very expensive and time consuming. Fast and easy DNA extractions can be performed by inhibitor binding by sorbent technology or by the use of chelating resin, but both methods deliver DNA of poor quality and further applications might be hindered due to compounds still present in the DNA solution. Even though for DNA barcoding standardized DNA extraction protocols can be used for a broad range of taxa, some groups are still left problematic. Especially for taxa containing high quantities of polysaccharides, mucopolysaccharides, polyphenols, resins, or other secondary metabolites, substances known for binding firmly to nucleic acids during DNA extraction procedure and/or interfering with subsequent reactions, specialized protocols were suggested (10–15). DNA isolation methods for small taxa, for example nematodes (16), tardigrades (17, 18), copepods (19), collemboles (20), or mites (21–23), as well as DNA isolation methods for fungi (24–26) or plants (12, 27–32) may help to recover DNA of sufficient quantity and quality. Besides the extraction of DNA, high-quality long term storage of the DNA samples, in order to conserve the genetic resources, verify the already existing results, or conduct further analyses, is challenging. Although in biological research millions of DNA samples are currently being processed and DNA and tissue banks were founded all around the world, the process of DNA degradation during storage is barely investigated and only few studies are available dealing with this subject (e.g., 33–38). Currently, there is no common sense about the optimal DNA storage conditions, but several commercial products are already available which allow storage of small amounts of dehydrated DNA at room temperature. These products are based on the natural principle of anhydrobiosis which can be found in Tardigrades using a mixture of dissolvable

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compounds, e.g., trehalose, that stabilize DNA for storage at room temperature (35). For the long-term storage of higher amounts of DNA, a combination of both appropriate preserving agents and low storage temperatures (−20°C or lower) is needed to minimize the loss of DNA quality during storage. In DNA barcoding, DNA extracts are used for amplification of a specific predefined gene region by using the technique of the polymerase chain reaction (PCR) which utilizes short, user-defined DNA sequences called oligonucleotide primers. In the first step, the DNA double helix is denaturized by heating into single-stranded template DNA, where the primers are able to bind then. The thermostable enzyme DNA Polymerase starts to extend the primers by adding single Deoxynucleotide triphosphates (dNTPs) producing new doublestranded DNA. This process is performed in a Thermocycler and has to be repeated several times to increase the number of the target fragments exponentially. The quality of the PCR products is commonly checked by agarose gel electrophoresis. Before sequencing, PCR products have to be purified to eliminate the remaining PCR ingredients. The use of suitable primers is essential for amplification success. Barcoding primers should correspond to rather conservative sites with low substitution rates to apply them to a broad range of taxa. Such “universal” primers amplifying an approximately 650-bp-long fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene were first defined by Folmer et al. (39) and then suggested as barcoding primers for the whole animal kingdom (40). In the course of time, it turned out that these primers are not applicable for all animal taxa and more and more group-specific ones were additionally suggested. In case of sponges (www.spongebarcoding. org), plants (41), or fungi (42), even new or additional barcoding regions were defined to get a resolution on species level. Although a tremendous variety of primers is available now, the design of new primers or the adjustment of existing primers remains still necessary for successful and effective DNA barcoding.

2. Materials 2.1. DNA Extraction

1. DNeasy Blood and Tissue Kit single columns and DNeasy 96 Blood and Tissue Kit (Qiagen): Buffers ATL, AL, AW1, AW2, AE, and Proteinase K are included in the kit. 2. NucleoSpin® Tissue Kit single columns and NucleoSpin® 96 Tissue Kit (Macherey-Nagel): Buffers T1, B1, B2, BW, B5, BE, PB, BQ1, and Proteinase K are included in the kit. 3. NucleoSpin® Plant II Kit single columns and NucleoSpin® 96 Plant II Kit (Macherey-Nagel): Buffers PL1, PC, PW1, PW2, PE, and RNase A are included in the kit (see Note 1).

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4. CTAB buffer: 100 ml of 1 M Tris–HCl (pH 8.0), 280 ml of 5 M NaCl, 40 ml of 0.5 M ethylene-diamine-tetraacetic-acid (EDTA), 20 g cetyltrimethyl-ammonium-bromide (CTAB). 5. TE buffer: 10 mM Tris–HCl, 1 mM EDTA pH 8.0. 6. Chloroform-isoamyl-alcohol: Chloroform:Isoamyl-alcohol, 24:1 (can be ordered at different companies). 2.2. DNA Preservation

1. 2 M trehalose stock solution: Dissolve 7.6 g trehalose (d-(+)Trehalose-dihydrate (Sigma Aldrich)) in 10 ml molecular water. 2. Qiasafe tubes and plates (Qiagen).

2.3. DNA Amplification

2.4. PCR Cleanup

1× TBE buffer: TBE is used as running buffer and for dissolving the agarose powder. To prepare the buffer, 10× TBE can be ordered (Rothiphorese 10× TBE buffer, Roth) and diluted to a 1× TBE buffer solution. Alternatively prepare one liter of 10× TBE buffer by mixing 108 g Tris, 55 g boric acid, and 7.4 g Na-EDTA in a beaker together with 500 ml demineralized water and heat for 20 min at 60°C (use a magnetic stir bar). Filter the buffer and transfer the solution to a bottle and fill up to 1 l. 1. NucleoSpin® Extract II Kit (Macherey-Nagel): Buffers NT3, NT, and NE are included in the kit. 2. Ethanol precipitation: Ethanol 100% and 70%, 3 M sodium acetate.

3. Methods 3.1. DNA Extraction: Source Material

Fresh material, if not immediately used for DNA extraction, should be directly frozen (−20°C or at lower temperatures) or fixed and preserved in 96% pure ethanol (large specimens can be subsampled) and stored at least at −20°C. Preserving agents, like DESS (43) or RNAlater (RNAlater RNA Stabilization Reagent, Qiagen), have also been proven to prevent DNA degradation during sample storage (see Note 2). Vascular plants, algae, and fungi may be better rapidly dried on silica gel and then stored in a dry, cool and dark place. Ancient material from museum collections exhibit dramatic DNA degradation (Fig. 1). Whenever possible, use recent material for DNA extraction.

3.2. DNA Extraction: Sample Preparation

1. Decontaminate workbench from any DNA using agents, like DNA Exitus Plus (BioChem) (see Note 3). 2. Wear new gloves. 3. Decontaminate all other tools (forceps, scalpels, etc.) from any DNA by flame (Bunsen burner) between processing each sample.

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Fig. 1. DNA extracts of Phalera bucephala (Lepidoptera) performed with Qiagen Blood and Tissue Kit on agarose gel. Dried specimens were taken from a museum collection collected in 2007 (1 ), 1971 (2 ), and 1935 (3 ). Number (1 ) contains DNA fragments up to 20 kb, whereas numbers (2 ) and (3 ) show dramatic DNA degradation with fragments between 100 and 300 bp.

4. Use 1.5-ml microcentrifuge tubes (e.g., Eppendorf ) for lysis (not included in commercially available kits); in case of plate extractions (96-well format), special lysis plates (deep-well plates) are provided (see Note 4). 5. Transfer a predefined (see DNA extraction protocols) amount of tissue into lysis tubes or deep-well plates (see Note 5). 6. Air dry EtOH-preserved tissue until alcohol is evaporated completely by placing the tubes with open caps in a Thermomixer

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at 40°C. Dried, fresh, or frozen material can be used directly for DNA extraction (see Note 6). 7. Clean workbench again before extraction procedure and wear new gloves. 8. Use filter tips and change pipette tips between each reagent. Do not touch the surface with tip. Keep pipette always in upright position. 3.3. DNA Extraction: Protocol Overview

For DNA isolation from animal and fungal samples, DNeasy Blood and Tissue Kit (Qiagen) (Subheadings 3.4 and 3.5) or NucleoSpin® Tissue Kit (Macherey-Nagel) (Subheadings 3.6 and 3.7) is recommended. The latter might be preferred especially for DNA extractions of arthropod samples. For plant samples, NucleoSpin® Plant II Kit (Macherey-Nagel) (Subheadings 3.8 and 3.9) achieves optimal DNA yields. All these kits are available in convenient single preparation as well as in 96-well plate format for high-throughput extractions. Alternatively, in case of mollusc, fungal, and algal taxa containing high amounts of mucopolysaccharides, better results might be achieved using the proposed CTAB protocol (Subheading 3.10) (see Note 7).

3.4. DNA Extraction: Protocol 1

DNA isolation from animal and fungal tissue with DNeasy Blood and Tissue Kit (Qiagen) single columns (see Note 8). 1. Adjust water bath or Thermomixer to 56°C (see Note 9). Add ethanol (96–100%; not provided with the kit) to buffers AW1 and AW2 as indicated on the bottles. 2. Place up to 25 mg of tissue in a 1.5-ml microcentrifuge tube (not provided). 3. Add 180 ml Buffer ATL and 20 ml Proteinase K and mix by inverting or vortexing. Briefly centrifuge samples at 3,000 × g. Incubate samples at 56°C for a few hours or overnight until tissue is lysed (see Note 10). 4. Vortex for 15 s. Briefly centrifuge samples at 3,000 × g. Add 200 ml Buffer AL to the lysate. 5. Immediately add 200 ml ethanol (96–100%), and mix by vortexing. Briefly centrifuge samples at 3,000 × g. 6. Transfer the mixture from step 5 into the DNeasy Mini spin column placed in a 2-ml collection tube (provided). Centrifuge at 6,000 × g for 1 min. Discard flow through and collection tube. 7. Place the DNeasy Mini spin column in a new 2-ml collection tube. Add 500 ml Buffer AW1 and centrifuge for 1 min at 6,000 × g. Discard flow through and collection tube. 8. Place the DNeasy Mini spin column in a new 2-ml collection tube. Add 500 ml Buffer AW2 and centrifuge for 3 min at 20,000 × g. Discard flow through and collection tube.

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9. Place the DNeasy Mini spin column in a clean 1.5-ml microcentrifuge tube (not provided) and add 100 ml Buffer AE directly onto the DNeasy membrane (see Note 11). Incubate at room temperature for 1 min, and then centrifuge for 1 min at 6,000 × g to elute DNA. 10. Repeat step 9 with the same microcentrifuge tube. A new microcentrifuge tube can be used for the second elution step to prevent dilution of the first eluate. 11. DNA should be immediately stored at −20°C. 3.5. DNA Extraction: Protocol 2

DNA isolation from animal and fungal tissue with DNeasy 96 Blood and Tissue Kit (Qiagen) (see Note 8). 1. Adjust water bath or Thermomixer to 56°C (see Note 9). Add ethanol (96–100%; not provided with the kit) to Buffers AL, AW1, and AW2 as indicated. If multichannel pipettes are used (recommended), sterilized reservoirs are required. 2. Place up to 20 mg of tissue in each collection microtube (96-well format, provided). 3. Add 180 ml Buffer ATL and 20 ml Proteinase K to each sample. Seal the collection microtubes properly with the provided cap strips. Mix by inversion. Briefly centrifuge up to 1,500 × g. Incubate samples at 56°C for a few hours or overnight until tissue is lysed (see Note 10). 4. Ensure that the microtubes are still properly sealed and mix by inversion for 15 s. Briefly centrifuge racks at 1,500 × g. Carefully remove the caps. Add 410 ml Buffer AL–ethanol to each sample. Seal collection microtubes with new cap strips and mix by inversion for 15 s. 5. Briefly centrifuge racks up to 1,500 × g. Place two DNeasy 96 plates on top of S-Blocks (provided). 6. Remove the first cap strip from the collection microtubes and carefully transfer the lysate to the DNeasy 96 plates. Continue with the next eight samples and so on until all samples are transferred. Seal DNeasy 96 plates with AirPore Tape Sheets (provided). Centrifuge for 10 min at 6,000 × g. 7. Remove the tape, and check that all of the lysate has passed through the membrane in each well of the DNeasy 96 plates. If lysate remains in any of the wells, centrifuge for further 10 min. 8. Remove the tape and carefully add 500 ml Buffer AW1 to each well. Seal each DNeasy 96 plate with a new AirPore Tape Sheet. Centrifuge for 5 min at 6,000 × g. 9. Remove the tape. Carefully add 500 ml Buffer AW2 to each well. Centrifuge for 15 min at 6,000 × g. Do not seal the plate

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with AirPore Tape Sheet in this step to allow evaporation of residual ethanol. 10. Place each DNeasy 96 plate in the correct orientation on a rack of Elution Microtubes RS (provided). 11. To elute the DNA, add 150 ml Buffer AE to each sample (see Note 11), and seal the DNeasy 96 plate with new AirPore Tape Sheet. Incubate for 1 min at room temperature (15–25°C). Centrifuge for 2 min at 6,000 × g. 12. Repeat step 11 with another 150 ml Buffer AE. Use appropriate cap strips (provided) to seal the Elution Microtubes RS for storage. 13. DNA should be immediately stored at −20°C. 3.6. DNA Extraction: Protocol 3

DNA isolation from animal and fungal tissue with NucleoSpin® Tissue Kit (Macherey-Nagel) single columns (see Note 8). 1. Prepare Buffer B3 by transferring buffer B1 into Buffer B2 (see Note 12). Dissolve Proteinase K (lyophilized) by adding the volume of Proteinase Buffer (PB) that is indicated on the Proteinase K label (see Note 13). Add appropriate volume (see label on Buffer B5 bottle) of ethanol (96–100%; not provided with the kit) to Buffer B5 before use. Adjust water bath or Thermomixer at 56°C (see Note 9). After tissue lysis, prepare a 70°C water bath and warm Buffer BE (elution buffer) to 70°C before use. 2. Place up to 25 mg of tissue in a 1.5-ml microcentrifuge tube (not provided). 3. Add 180 ml of Buffer T1 and 25 ml of Proteinase K to each sample and mix by inverting or vortexing. Briefly centrifuge samples at 3,000 × g. Incubate samples at 56°C for a few hours or overnight until tissue is lysed (see Note 10). 4. Vortex for 15 s. Briefly centrifuge samples at 3,000 × g. Add 200 ml of Buffer B3. Vortex and incubate at 70°C for 10 min. 5. Briefly centrifuge samples at 3,000 × g. Add 210 ml of 96–100% ethanol and vortex immediately. 6. Briefly centrifuge samples at 3,000 × g. Transfer the mixture from step 5 into the NucleoSpin column placed in a 2-ml collection tube (provided). Centrifuge at 11,000 × g for 1 min. Discard flow through. 7. Add 500 ml of Buffer BW to the spin column. Centrifuge at 11,000 × g for 1 min. Discard flow through. 8. Add 600 ml of Buffer B5 to the spin column. Centrifuge at 11,000 × g for 1 min. Discard the flow through. Centrifuge again at 11,000 × g for 1 min to remove the residual Buffer B5.

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9. Place the NucleoSpin column in a clean 1.5-ml microcentrifuge tube (not provided) and pipette 100 ml of Buffer BE (warmed to 70°C) onto the NucleoSpin membrane (do not touch the membrane) (see Note 11). Incubate at room temperature for 1 min, and then centrifuge at 11,000 × g for 1 min to elute DNA. 10. Repeat step 9 with the same microcentrifuge tube. A new microcentrifuge tube can be used for the second elution step to prevent dilution of the first eluate. 11. DNA should be immediately stored at −20°C. 3.7. DNA Extraction: Protocol 4

DNA isolation from animal and fungal tissue with NucleoSpin® 96 Tissue Kit (Macherey-Nagel) (see Note 8). 1. Dissolve Proteinase K (lyophilized) by adding the volume of Proteinase Buffer (PB) that is indicated on the Proteinase K label and store at −20°C (see Note 13). Add appropriate volume (see label on Buffer B5 bottle) of ethanol (96–100%; not provided with the kit) to Buffer B5 before use. Adjust water bath or Thermomixer at 56°C (see Note 9). After tissue lysis, preheat incubator at 70°C and warm Buffer BE (elution buffer) to 70°C before use. 2. Place up to 20 mg of tissue into each well of a Round-well Block (provided). 3. Add 180 ml Buffer T1 and 25 ml of Proteinase K to each sample. Seal wells properly with cap strips and mix by inverting for 10–15 s. Spin briefly for 15 s at 1,500 × g. Incubate samples at 56°C for a few hours or overnight until tissue is lysed (see Note 10). 4. Ensure that the microtubes are still properly sealed and centrifuge the Round-well Block for 15 s at 1,500 × g. Carefully remove cap strips. Add 200 ml of Buffer BQ1 and 200 ml of 96–100% ethanol to each sample. Close the wells with new cap strips and mix by inversion for 10–15 s. Centrifuge racks for 10 s at 1,500 × g. 5. Place each NucleoSpin plate onto a MN Square-well Block (provided). 6. Remove the first cap strip from the first eight wells and carefully transfer the lysate to the NucleoSpin plates. Continue with the next eight samples and so on until all samples are transferred. Seal each NucleoSpin plate with adhesive PE foil (provided). Centrifuge for 10 min at 5,600 × g. 7. Remove foil, and check that all of the lysate has passed through the membrane in each well of the NucleoSpin plates. If lysate remains in any of the wells, centrifuge for further 10 min.

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8. Carefully add 500 ml of Buffer BW to each well. Seal each plate with a new adhesive PE foil. Centrifuge for 2 min at 5,600 × g. 9. Remove adhesive PE foil. Carefully add 700 ml of Buffer B5 to each well. Seal the plate with a new adhesive PE foil. Centrifuge for 4 min at 5,600 × g. 10. Remove adhesive PE foil. Place NucleoSpin plates on an opened rack with tube strips and incubate for 10 min at 70°C in an incubator to evaporate residual ethanol. 11. To elute DNA, dispense 100 ml of prewarmed (70°C) Buffer BE (elution buffer) to each well directly onto the membrane of the NucleoSpin plates (see Note 11). Incubate at room temperature for 1 min. Centrifuge for 2 min at 5,600 × g. 12. Repeat step 11 with another 100 ml Buffer BE. 13. Remove the NucleoSpin plate and seal tube strips. DNA should be immediately stored at −20°C. 3.8. DNA Extraction: Protocol 5

DNA isolation from plant tissue with NucleoSpin® Plant II Kit (Macherey-Nagel) single columns (see Note 8). 1. Add appropriate volume (see label on Buffer PW2 bottle) of ethanol (96–100%; not provided with the kit) to Buffer PW2 before use. Dissolve RNase A (lyophilized) by adding the volume of molecular water that is indicated on the RNase A label and store at −20°C (see Note 14). Adjust water bath or Thermomixer at 65°C (see Note 9). After tissue lysis, preheat incubator at 70°C and warm Buffer PE (elution buffer) to 70°C before use. 2. Homogenize 50 mg (up to 100 mg) wet-weight or 10 mg (up to 20 mg) dry-weight (lyophilized) plant material (see Note 15). 3. Transfer the resulting powder to a new 1.5-ml microcentrifuge tube (not provided) and add 400 ml Buffer PL1. Vortex the mixture thoroughly. Add 10 ml RNase A solution and mix sample thoroughly. Incubate the suspension for 10 min at 65°C. 4. Briefly centrifuge samples at 3,000 × g. Place a NucleoSpin® Filter column (violet ring) into a 2-ml collection tube and load the lysate onto the column. Centrifuge for 2 min at 11,000 × g, collect the clear flow through (see Note 16), and discard the NucleoSpin® Filter. 5. Add 450 ml Buffer PC and mix thoroughly by vortexing. 6. Briefly centrifuge samples at 3,000 × g. Place a NucleoSpin® Plant II Column (green ring) into a new 2-ml collection tube and load a maximum of 700 ml of the sample. Centrifuge for 1 min at 11,000 × g and discard flow through (see Note 17).

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7. Add 400 ml Buffer PW1 to the NucleoSpin® Plant II Column. Centrifuge for 1 min at 11,000 × g and discard flow through. 8. Add 700 ml Buffer PW2 to the NucleoSpin® Plant II Column. Centrifuge for 1 min at 11,000 × g and discard flow through. 9. Add another 200 ml Buffer PW2 to the NucleoSpin® Plant II Column. Centrifuge for 2 min at 11,000 × g in order to remove wash buffer and dry the silica membrane completely. 10. Place the NucleoSpin® Plant II Column into a new 1.5-ml microcentrifuge tube (not provided). Pipette 50 ml Buffer PE (preheated to 70°C) onto the membrane. Incubate the NucleoSpin® Plant II Column for 5 min at 70°C. Centrifuge for 1 min at 11,000 × g to elute the DNA. 11. Repeat step 10 with another 50 ml Buffer PE (preheated to 70°C) and elute into the same tube. 12. DNA should be immediately stored at −20°C. 3.9. DNA Extraction: Protocol 6

DNA isolation from plant tissue with NucleoSpin® 96 Plant II Kit (Macherey-Nagel) (see Note 8). 1. Add appropriate volume (see label on Buffer PW2 bottle) of ethanol (96–100%; not provided with the kit) to Buffer PW2 before use. Dissolve RNase A (lyophilized) by adding the volume of molecular water that is indicated on the RNase A label and store at −20°C (see Note 14). Adjust water bath or Thermomixer at 65°C (see Note 9). After tissue lysis, preheat incubator at 70°C and warm Buffer PE (elution buffer) to 70°C before use. 2. Homogenize 50 mg (up to 100 mg) wet-weight or 10 mg (up to 20 mg) dry-weight (lyophilized) plant material (see Note 15) in each tube of the tube strips. 3. Add 500 ml Buffer PL1 and 10 ml RNase A to each sample. Close tubes using cap strips (provided). Mix vigorously by shaking for 15–30 s. Centrifuge briefly for 30 s at 1,500 × g. Incubate samples at 65°C for 30 min. 4. Centrifuge the samples for 20 min at 5,600 × g. Remove cap strips. 5. Predispense 450 ml Binding Buffer PC to each well of an MN Square-well Block. Add 400 ml cleared lysate of each sample and mix by repeated pipetting up and down. Mix at least three times. 6. Place NucleoSpin® Plant II Binding Plate on an MN Squarewell Block. Transfer samples from the previous step into the wells of the NucleoSpin® Plant II Binding Plate. Do not moisten the rims of the individual wells while dispensing the samples. 7. Place the NucleoSpin® Plant II Binding Plate stacked on an MN Square-well Block in the rotor buckets. Centrifuge at 5,600 × g for 5 min.

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8. Add 400 ml PW1 to each well of the NucleoSpin® Plant II Binding Plate. Optional: Seal plate with a gas-permeable foil. Centrifuge again at 5,600 × g for 2 min. Place NucleoSpin® Plant II Binding Plate on a new MN Square-well Block. 9. Add 700 ml PW2 to each well of the NucleoSpin® Plant II Binding Plate. Optional: Seal plate with a gas-permeable foil. Centrifuge again at 5,600 × g for 2 min. 10. Add 700 ml PW2 to each well of the NucleoSpin® Plant II Binding Plate. Optional: Seal plate with a gas-permeable foil. Centrifuge again at 5,600 × g for 10 min for complete removal of residual Buffer PW2. 11. Place NucleoSpin® Plant II Binding Plate on the rack with tube strips. Dispense 100 ml Buffer PE (preheated 70°C) directly onto the membrane of each well of the NucleoSpin® Plant II Binding Plate. Incubate at room temperature for 2 min. Centrifuge at 5,600 × g for 2 min. 12. Repeat step 10 with another 100 ml Buffer PE (preheated to 70°C) and elute into the same rack with tube strips. 13. DNA should be immediately stored at −20°C. 3.10. DNA Extraction: Protocol 7

DNA isolation from tissue containing high amounts of mucopolysaccharides with CTAB method. 1. Adjust water bath or Thermomixer at 55°C (see Note 9). Mark and precool 1.5-ml microcentrifuge tubes containing 25 ml 3 M ammonium acetate + 600 ml 70% ethanol per sample at 4°C. Mark another two sets of tubes according to the number of samples. Precool 70% ethanol. 2. Place tissue sample to one set of marked tubes. Ground sample (5–20 mg tissue) if necessary or let the residual ethanol evaporate. 3. Add 300 ml CTAB buffer and 0.6 ml b-mercaptoethanol per sample (see Note 18). Perform this step under a fume hood. 4. Add 10 ml Proteinase K (20 mg/ml, Qiagen) to each sample and mix carefully. 5. Incubate samples at 55°C for a few hours or overnight until tissue is lysed (see Note 10). 6. Add 300 ml chloroform-isoamyl-alcohol (24:1) and mix well by shaking tubes for 2 min. Perform this step under a fume hood. Proteins are precipitated now. 7. Centrifuge for 10 min at 11,000 × g. Pipette the supernatant into another set of clean tubes. 8. Add another 300 ml chloroform-isoamyl-alcohol (24:1) to the new set of tubes including the supernatant from step 7 and mix

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well by shaking tubes for 2 min. Perform this step under a fume hood. Remaining proteins are precipitated now. 9. Centrifuge for 10 min at 12,000 × g. Pipette the supernatant to the set of clean tubes containing cold 25 ml 3 M ammonium acetate + 600 ml 70% ethanol. The supernatant includes DNA which is precipitated in this step. 10. Centrifuge for 10 min at 12,000 × g. Pour or pipette off the liquid, being careful not to touch or lose the DNA pellet (see Note 19). 11. Add 250 ml cold 70% ethanol and mix to wash the DNA pellet. 12. Centrifuge for 10 min at 12,000 × g. 13. Pour or pipette off the liquid. Dry pellet in the incubator for 5–10 min (at 60°C). 14. Dissolve the pellet in 50 ml TE buffer. 15. DNA should be immediately stored at −20°C. 3.11. DNA Preservation: Protocol 1

According to the DNA extraction protocols, exclusively use buffers to elute or dissolve DNA (see Note 20). For storage, DNA isolates ought to be portioned into two (or more) aliquots. One aliquot serves as backup for long-term storage at −80°C. The other aliquot(s) can be kept as working solution at −20°C to be used for PCR amplification (see Note 21). The best method to preserve high DNA quality of the backup aliquot is the use of QIAsafe DNA Tubes (Qiagen), which contain a mixture of dissolvable compounds that stabilize DNA (see Note 22). For sample storage, proceed according to the following steps. 1. Pipette up to 50 ml of the DNA solution (not more than 30 mg DNA) on the colored DNA-protecting matrix of the QIAsafe DNA Tubes. 2. Dry samples with a vacuum concentrator (1 h at 55°C for 20 ml DNA solution) or under a laminar flow hood at room temperature (12 h for 20 ml of DNA solution). 3. Seal completely dried samples and preferably store QIAsafe DNA Tubes at −80°C (see Note 23). 4. To recover DNA, dissolve pellet of dried protection matrix including DNA with appropriate volume of molecular water. DNA solution can immediately be used for PCR or other applications.

3.12. DNA Preservation: Protocol 2

QIAsafe DNA Tubes are relatively cost intensive. Therefore, we suggest a further professional method to store DNA using the preserving agent trehalose. 1. Transfer 90 ml DNA extract to sterile and temperature-stable storage tubes (e.g., Rotilabo®-microcentrifuge tubes, Roth).

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2. Add 10 ml of trehalose stock solution (2 M) to obtain a final concentration of 200 mM in the DNA sample (see Note 24). 3. Dry samples with a vacuum concentrator at 55°C (this may last for a few hours). 4. Store sealed samples at −80°C (see Note 25). 5. To recover DNA, dissolve pellet with appropriate volume of molecular water. DNA solution can immediately be used for PCR or other applications. 3.13. DNA Amplification: PCR Ingredients

1. DNA Polymerase: Recombinant Taq DNA Polymerase (e.g., Qiagen) is commonly used for standard PCR. It is a thermostable enzyme of the thermophilic bacterium Thermus aquaticus and is, therefore, able to synthesize DNA at high temperatures (see Note 26). Usually, 0.025 U of Taq DNA Polymerase are used per ml of the PCR reaction. Hot Start Polymerases can be used to prevent the amplification of unspecific PCR products before the PCR program is started. They are inactive at lower temperatures and are activated during the first heating step of the PCR program. 2. PCR buffer: For optimal DNA Polymerase reaction activity, PCR buffers are used containing Tris–HCl, KCl, and, optional, MgCl2. Buffers are provided by the supplier together with Taq Polymerase. It is important to use Polymerase and PCR buffer from the same manufacturer. 3. Oligonucleotide primers: PCR primers are short, singlestranded DNA fragments (usually, 20–30 nucleotides). PCR requires one forward and one reverse primer to assign the favored fragment of the DNA. Primers are usually delivered in desalted mode. Resuspend primers in molecular water to a stock concentration of 100 pmol/ml; prepare aliquots of working solutions with a concentration of 10 pmol/ml ready to use for PCR. There should be a surplus of primers in the reaction mix, but too much of them may lead to unspecific reactions. For standard PCR, 0.5 ml of each primer working solution (10 pmol/ml) is enough. For primer design, see Note 27. 4. Deoxynucleotide triphosphates (dNTPs): dNTPs (dATP, dTTP, dGTP, and dCTP) are the nucleotide bases added by the DNA Polymerase during synthesis of the template strand. They are available as single ingredients or as a dNTP mix (e.g., Fermentas). There should always be a slight surplus of dNTPs in the reaction mix. For PCR, a final concentration of 2 mM dNTPs (which means 2 mM of each type of nucleotide!) is applicable. In case of the nucleotide premix (all nucleotides in a total concentration of 10 mM), the solution has just to be diluted to the desired concentration. In case of single nucleotides, add 20 ml (100 mM stocks) of each dNTP to 920 ml molecular water.

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5. Additives: Additives, like MgCl2, trehalose, DMSO, Q-solution, etc., can enhance PCR efficiency (see Note 28). Use additives only if standard protocols do not work. Too high concentrations of MgCl2, for instance, increase the amount of unspecific products due to unspecific amplification of the Polymerase. 6. Molecular water: Use only ultra pure and nuclease-free water for PCR. Water is used to fill the mix of ingredients up to the desired volume, which is normally 10–25 ml. 7. Template DNA: This is the original genomic DNA material. Use 1–2 ml DNA solution (obtained from extraction) with a concentration between 20 and 100 ng/ml. Usually, PCR also works well with lower concentrations (below 2 ng/ml) (see Note 29). 3.14. DNA Amplification: Principle Steps

PCR amplification is carried out in a Thermocycler using a specific temperature profile. It involves initial denaturation of the template DNA, followed by a specific number of cycles, including denaturation, primer annealing, and elongation steps. The program is finished by an extended final elongation step (see Note 30). 1. Initial denaturation: Melting of double-stranded DNA in two single-stranded templates by disrupting the hydrogen bonds between complementary nucleotides. This step is usually performed at a temperature of 94°C for about 5 min. If the template DNA is GC rich, the interval should be extended up to 10 min. Heating the lid is recommended and normally an option of every Thermocycler. 2. Denaturation: Similar to the initial denaturation, this step leads to melting of the double-stranded DNA into single strands for primer annealing. Amplified DNA with high GC content needs increased denaturation time (3–4 min). 3. Annealing: In most cases, temperatures between 50 and 65°C allow successful annealing of primers to the single-template DNA strands. Typically, the optimal annealing temperature (Ta) is 3–5°C below the melting temperature of the primers (Tm). Tm can be calculated by several computer programs (see primer design below). If nonspecific PCR products are produced in addition to the desired product, temperature can be optimized by stepwise raising in increments of 1–2°C. 4. Elongation: In this step, the DNA Polymerase synthesizes a new DNA strand complementary to the template strand by adding dNTPs. The optimal elongation temperature is dependent of the Polymerase itself and the length of the desired fragment. In case of Taq DNA Polymerase, the highest synthesis rates can be performed at 70–75°C. For fragments up to 1,000 bp, optimal elongation time is between 1 and 2 min.

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For longer fragments, more elongation time is needed (vice versa for smaller fragments). 5. Number of cycles: Now, the steps 2–4 are repeated several times (cycles). The number of cycles depends on the amount of template DNA. If the initial DNA quantity is low, up to 40 cycles can be performed. For higher amounts of template, 30–35 cycles may last. 6. Final elongation: After the last PCR cycle, a final elongation is performed to ensure that all remaining single DNA strands are fully extended. It is usually performed at 72°C for 5–10 min. 7. Cooling (optional): After the final elongation step, samples can remain in the Thermocycler if reactions are performed overnight. For cooling overnight, use a temperature of 15°C. This temperature neither damages PCR products nor strains the heating block too much. Subsequently to amplification, the PCR products can be stored for a while in the fridge (4°C) until further processing. 3.15. DNA Amplification: PCR Performance

It is recommended to prepare a so-called master mix for all samples that should be processed. The master mix contains all required ingredients, except the template DNA (Subheadings 3.16 and 3.17, see also Note 31). Volumes of ingredients are calculated according to the number of processed samples including a positive and a negative control (see Note 32) plus about 5–10% more just to make sure that it is enough. (It is very annoying if there is no master mix left for the last few samples!) For PCR preparation, notice the following steps: 1. Select samples for PCR. Calculate volumes of required ingredients according to the protocol. 2. Wear new gloves and use filter tips for pipetting steps to avoid contamination. 3. Thaw required ingredients and DNA samples. Vortex all ingredients, except DNA samples. Briefly centrifuge all ingredients and DNA samples before opening. Keep everything on ice. 4. Prepare master mix with all ingredients, except Taq DNA Polymerase. Start with water or buffer first. Use PCR form as checklist (make check marks). Find below protocols for master mix preparation. 5. Transfer 1–2 ml of DNA template to 0.2-ml PCR reaction tubes (single tubes, 8-stripes or 96-well PCR plates, dependent on the number of samples). 6. Take Taq DNA Polymerase out of the freezer and centrifuge briefly. Add Polymerase to master mix and pipette up and down carefully to mix (put Polymerase immediately back to the freezer!). Dispense master mix to PCR reaction tubes (see Note 33).

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7. Cap PCR tubes properly (in case of plates, seal with foil) and mark for identification (use thermostable markers, e.g., Stabilo OH Pen). 8. Briefly centrifuge the PCR tubes, place them into the Thermocycler, and start the required program. After PCR, check products on agarose gel (see Subheading 3.20). 3.16. DNA Amplification: PCR Master Mix Protocol 1

Protocol for one sample using Taq DNA Polymerase (e.g., Qiagen) with a 25 ml PCR reaction volume; dispense 24 ml of the master mix to 1 ml of the template DNA (see Note 34): 1. Molecular-grade water: 15.875 ml. 2. 10× PCR buffer: 2.5 ml. 3. MgCl2: 2.0 ml. 4. dNTPs, 2 mM each: 2.5 ml. 5. Primer forward, 10 pmol/ml: 0.5 ml. 6. Primer reverse, 10 pmol/ml: 0.5 ml. 7. Taq Polymerase 5 U/ml: 0.125 ml. 8. DNA: 1.0 ml.

3.17. DNA Amplification: PCR Master Mix Protocol 2

Protocol for one sample using Hot Start DNA Polymerase (e.g., Phire-Polymerase, New England BioLabs) with a 20 ml PCR reaction volume; dispense 19 ml of the master mix to 1 ml of the template DNA (DMSO is provided by the supplier of the Polymerase). 1. Molecular water: 11.2 ml. 2. 5× PCR buffer: 4.0 ml. 3. DMSO: 1.0 ml. 4. dNTPs, 10 mM each: 0.4 ml. 5. Primer forward, 10 pmol/ml: 0.5 ml. 6. Primer reverse, 10 pmol/ml: 0.5 ml. 7. Phire-Polymerase: 3.6 ml. 8. DNA: 1.0 ml.

3.18. DNA Amplification: PCR Temperature Scheme Protocol 1

Standard temperature profile using Taq DNA Polymerase. 1. Initial step: 94°C—2 min. 2. Denaturation: 94°C—30 s. 3. Annealing: 50°C—30 s. 4. Elongation: 72°C—1 min. 5. Cycles: Repeat steps 2–4 for 35 times. 6. Final elongation: 72°C—10 min. 7. Cooling: 15°C—as long as required (often called “forever” in programs of Thermocyclers).

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3.19. DNA Amplification: PCR Temperature Scheme Protocol 2

Temperature profile using Hot Start Polymerase (see Note 35). 1. Initial step: 98°C—30 s. 2. Denaturation: 98°C—5 s. 3. Annealing: 65°C—5 s. 4. Elongation: 72°C—35 s. 5. Cycles: Repeat steps 2–4 for 35 times. 6. Final elongation: 72°C—1 min. 7. Cooling: 15°C—as long as required (often called “forever” in programs of Thermocyclers).

3.20. PCR Product Quality Control by Agarose Gel Electrophoresis (see Note 36)

1. Prepare loading dye and Molecular Size Marker (100 bp DNA Ladder Plus, Fermentas) according to the manufacturer’s instructions. Loading dye is used in 1× concentration in this protocol. 2. Prepare the tray with appropriate combs. 3. In case of a usual 100-ml gel, weigh 1.0 g agarose powder and add 100 ml 1× TBE buffer. Boil the mixture in a microwave until the agarose powder is completely dissolved (see Note 37). Add 2 ml (or one drop) ethidiumbromide or 10 ml GelRed and shake carefully. Immediately pour the mix to the prepared tray and wait until the agarose gel is solid which takes about half an hour. 4. Apply the gel to an adequate electrophoresis chamber filled with 1× TBE buffer. Gel should be completely dipped. Remove the combs. Mix 2 ml of each PCR product with 2 ml loading dye (prepared in a microtiter plate according to the number of samples) and pipette up and down a few times to mix. Load the PCR samples into the pockets (see Note 38). 5. Connect voltage (90 V) and let samples run for about 30 min. Afterwards, the double-stranded PCR products can be viewed in ultraviolet light. Take a photo to select samples for the cleanup. Sharp bands indicate successful amplification of the desired DNA fragment (Fig. 2). When PCR fails, no bands are present. In case of suspicious PCR results, see PCR troubleshooting in Note 39.

3.21. PCR Cleanup: ExoSAP-It

PCR purification eliminates the remaining PCR ingredients and single-stranded DNA fragments which may inhibit the adjacent sequencing reaction. Cleanup can be carried out in three different ways: enzymatic digestion (this section), ethanol precipitation (Subheading 3.22), or via commercial kits (Subheading 3.23) that are based on column methods (similar to the extraction methods of Qiagen and Macherey-Nagel). After the cleanup, the PCR products are ready to use for sequencing reactions.

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Fig. 2. Amplified COI fragments on agarose gel. Lanes 1–4 show very intense and sharp PCR products. In lanes 5–8, DNA amplification failed, and only unconsumed primers are visible.

Enzymatic digestion using ExoSAP-It (GE Healthcare, formerly Amersham Biosciences) (see Note 40): 1. Mark the appropriate number of tubes. Do cleanup of only the samples that worked well in the PCR reaction. Transfer 5 ml of each of the selected PCR products to new PCR tubes (single tubes, 8-stripes, or plates). 2. To clean up 5 ml of PCR product, 1.0 ml molecular water and 0.5 ml ExoSAP-It are needed. Produce a master mix of water and ExoSAP-It according to the number of samples plus about 5–10% more just to make sure that it is enough. 3. Mix 5 ml PCR product with 1.5 ml master mix, then place it to the Thermocycler, and start the program with the following temperature scheme: 37°C—40 min. 80°C—15 min. 15°C—as long as required. In the first step, Exonuclease I degrades residual single-stranded DNA and primers. Shrimp Alkaline Phosphatase hydrolyzes remaining dNTPs. Those ingredients would otherwise interfere with the sequencing reaction. In the second step, ExoSAP-It itself is inactivated. After this procedure, the cleaned up PCR product can be directly used for sequencing reaction (see Note 41). 3.22. PCR Cleanup: NucleoSpin® Extract II Kit

Column-based method using NucleoSpin® Extract II Kit (Macherey-Nagel): 1. Dilute buffer NT3 with the correct amount of ethanol (96–100%). Mark the appropriate number of 1.5-ml microcentrifuge tubes (not provided).

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2. For sample volumes of