Transcript 2. Prepare your sample

KEYS Lab Training
– DNA Barcoding: Identification of Species
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Pipetting: Measuring Volumes Accurately
DNA Extraction
PCR and Gel Electrophoresis
Sequencing and Analysis
– Protein Profile: Evolutionary Relationships
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Protein Extraction
Determine Protein Concentrations
SDS-PAGE
Stain Protein Gels and Analyze
THE INS AND OUTS OF PIPETTING
Push Button
Tip ejector button
Volume Adjustment Knob
• First Stop: Measurement
• Second Stop: Expel volume
Sizes & Volumes
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P10 Measures 0.5-10 ml
P20: Measures 2-20 ml
P200: Measures 20-200ml
P1000: Measures 200-1000ml
Tips & Tip Ejectors
• Match the tip to the Pipet
MEASURING VOLUMES
DNA Sequence Data is Obtained for
Genetic Research
Obtain DNA-bearing
tissue samples:
blood , saliva, hair
follicles, feathers,
scales
Extract DNA
from Cells
Sequence DNA
Identify
matching DNA
sequence(s) in
databases
TTCAACAACAGGCCCAC
TTCACCAACAGGCCCAC
TTCATCTACAGCCCCAC
…TTCACCAACAGGCCCACA…
From Sample to Sequence
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Sample
Extract the DNA
Amplify R.O.I.
Ensure amplification ok
Analyze DNA
– Restriction analysis
– Hybridization
– Sequencing
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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…
Fish DNAbarcode Primers
Primer mix: 2 forward, 2 reverse
•5’- TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC-3’
•5’- TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC-3’
•5’- CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAGAA-3’
•5’- CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA-3’
From Sequence to Conclusion
PCR is like DNA replication
• DNA Replication
• Polymerase Chain Reaction
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Nuclear DNA
DNA polymerases
Helicase
Primers (from Primase)
dNTPs
Sample DNA (from fish)
DNA polymerase
Heat
Primers (specific to COI gene)
dNTPs
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
The Power of PCR
View the animation at http://www.dnalc.org/resources/animations/pcr.html
(may require Flash player or Shockwave player)
http://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553
Fish DNA extraction for PCR
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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.
PCR Ingredients
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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
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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 2-4 30 times
72°C 10 min-final extension
Agarose Gel Electrophoresis
Molecular Weight
Standard
(DNA of Known Sizes)
Lanes:
2000 bp
1000 bp
750 bp
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Samples of DNA
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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
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
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
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
http://oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate.html
Agarose Gel Electrophoresis
• 1. Prepare agarose gel
Power Supply
• 2. Prepare your sample
• 3. Load your sample
Voltage/Amps
Electrodes
Red = Positive
Black = Negative
• 4. Run gel
• 5. Stain & view gel
Gel Box with Gel
http://bristoluniversityfacultyofscience.blogspot.com/2010/06/tanias-tales-from-lab-part-3.html
Agarose Gel Electrophoresis
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1. Prepare agarose gel
2. Prepare your sample
3. Load your sample
4. Run gel
5. Stain & view the gel
Ethidium Bromide & UV Light
Molecular
Weight
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Samples
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2000bp
1000bp
500bp
Separating pieces of DNA
based on size
Black & White image of UV
Fast Blast
http://scienceblogs.com/moleculeoftheday/2/10/ethidium_glowing_dna.php http://www.vernier.com/biotech/wht-dbs.html
http://www.vernier.com/biotech/wht-dbs.html
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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
http://www.scq.ubc.ca/genome-projects-uncovering-the-blueprints-of-biology/
Genetic Researchers Developed
Primers for Barcoding
Pool COI-2:
mammals,
and insects
Pool COI-3:
fish
Ivanova et al. 2007. Universal primer cocktails for fish barcoding. Mol Ecol Notes.