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KEYS Lab Training DNA Barcoding: Identification of Species
Pipetting: Measuring Volumes Accurately DNA Extraction PCR and Gel Electrophoresis Sequencing and Analysis Protein Profile: Evolutionary Relationships Protein Extraction Determine Protein Concentrations SDS-PAGE Stain Protein Gels and Analyze
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THE INS AND OUTS OF PIPETTING
Push Button First Stop: Measurement Second Stop: Expel volume Sizes & Volumes P10 Measures ml P20: Measures 2-20 ml P200: Measures ml P1000: Measures ml Tips & Tip Ejectors Match the tip to the Pipet Tip ejector button Volume Adjustment Knob
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Measuring Volumes
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DNA Sequence Data is Obtained for Genetic Research
Extract DNA from Cells Obtain DNA-bearing tissue samples: blood , saliva, hair follicles, feathers, scales Identify matching DNA sequence(s) in databases Sequence DNA …TTCACCAACAGGCCCACA… TTCAACAACAGGCCCAC TTCACCAACAGGCCCAC TTCATCTACAGCCCCAC
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From Sample to Sequence
Extract the DNA Amplify R.O.I. Ensure amplification ok Analyze DNA Restriction analysis Hybridization Sequencing Various Sushi Fish Dilution Buffer cracks cells Release Cores pick DNA Phire Animal Tissue Direct PCR Kit uses primers to amplify DNA R.O.I. Agarose Gel Electrophoresis DNA Sequencing …TTCACCAACAGGCCCACA…
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Fish DNAbarcode Primers
Primer mix: 2 forward, 2 reverse 5’- TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC-3’ 5’- TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC-3’ 5’- CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAGAA-3’ 5’- CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA-3’
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From Sequence to Conclusion
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PCR is like DNA replication
Nuclear DNA DNA polymerases Helicase Primers (from Primase) dNTPs Polymerase Chain Reaction Sample DNA (from fish) DNA polymerase Heat Primers (specific to COI gene) dNTPs
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PCR Ingredients 1. DNA “template” Your purified DNA sample
2. DNA Polymerase Special DNA polymerase enzyme that is heat stable 3. Deoxynucleotides (dNTPs) Building blocks of DNA 4. Primers Small pieces of DNA that match the flanks of your gene or DNA region of interest 5. Buffer and water Environment necessary for DNA Polymerase to work; mimics conditions in nucleus
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(may require Flash player or Shockwave player)
The Power of PCR View the animation at (may require Flash player or Shockwave player)
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Fish DNA extraction for PCR
Add 20 µl of Dilution Buffer to a 0.2 ml tube. Place your fish sample on a clean weigh boat, or other clean surface. Remove the protective cap of the Uni-Core tool. Gently push the cutting edge downward into the sample, rotating gently. Do not press the plunger while cutting. Lift the tool from the sample, position tool over tube and press the plunger so that the tissue drops directly into the dilution solution (make sure that you can see the sample in the solution). Add 0.5 µl of DNA release solution and vortex briefly, then spin down the solution (make sure the tissue is covered). Incubate 2-5 minutes at room temperature, then 2 minutes at 98°C. Spin down the tissue and move supernatant to a clean 0.65 ml tube.
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PCR Ingredients PCR Reaction Label top of tube
Add 20.5 ml of nuclease free H20 Add 25 ml of 2X Phire PCR Buffer Add 2.5 ml of DNA sample in Dilution buffer Add 2 ml of primer mix (both forward and reverse) Add 1 ml of Phire Hot Start DNA polymerase PCR Reaction 98°C 5 min-denaturation of all DNA in tube 98°C 5 sec-denaturation 52°C 20 sec-anneal 72 °C 20 sec-extension Repeat steps times 72°C 10 min-final extension
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Agarose Gel Electrophoresis
Molecular Weight Standard (DNA of Known Sizes) Samples of DNA Lanes: 2000 bp 1000 bp 750 bp
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Agarose Gel Electrophoresis
1. Make the agarose gel: prepare TAE and agarose 2. Prepare your sample: mix 10ul DNA with Loading Dye 3. Load your sample on the gel 4. Run the gel 5. Stain and view the gel
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Agarose Gel Electrophoresis
1. Prepare agarose gel 2. Prepare your sample 3. Load your sample 4. Run gel 5. Stain & view gel Agarose: from agar (seaweed) Melt the agarose and pour into a form or gel mold Small wells in the top of the gel for DNA samples Start at o minutes, run to 2:44
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Agarose Gel Electrophoresis
1. Prepare agarose gel 2. Prepare your sample 3. Load your sample 4. Run gel 5. Stain & view gel 10 mL DNA Loading Buffer 6X concentrated Color Glycerol
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Agarose Gel Electrophoresis
1. Prepare agarose gel 2. Prepare your sample 3. Load your sample on the gel 4. Run gel 5. Stain & view gel Start at o min, run to 2:52
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Agarose Gel Electrophoresis
1. Prepare agarose gel 2. Prepare your sample 3. Load your sample 4. Run gel 5. Stain & view gel Power Supply Voltage/Amps Electrodes Red = Positive Black = Negative Gel Box with Gel
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Agarose Gel Electrophoresis
Ethidium Bromide & UV Light 1. Prepare agarose gel 2. Prepare your sample 3. Load your sample 4. Run gel 5. Stain & view the gel Molecular Weight 500bp 1000bp 2000bp Samples Black & White image of UV Fast Blast Separating pieces of DNA based on size
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DNA Sequencing 1. DNA “template” Your PCR fragment, purified
2. Taq Polymerase Heat-stable DNA polymerase 3. Deoxynucleotides (dNTPs) and Dideoxynucleotides Building blocks of DNA; regular and altered 4. Primers Specific for your gene of interest 5. Buffer and water
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Genetic Researchers Developed Primers for Barcoding
Pool COI-2: mammals, and insects Pool COI-3: fish Ivanova et al Universal primer cocktails for fish barcoding. Mol Ecol Notes.
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