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miniPCR Shark Attack Lab
Science for everyone, everywhere miniPCR Shark Attack Lab Introduction to DNA fingerprinting V1.0 October 2016 © 2016 by Amplyus, all rights reserved
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Bridging science and education
Sebastian Kraves, PhD Zeke Alvarez Saavedra, PhD 24/7 support by our PhD Team (781) 990-8PCR
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Shark Attack Lab Engaging scenario DNA fingerprinting
Single class period Optimized for Electrophoresis system and transilluminator
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Genetic variation
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We’re 99.9% identical in our DNA
Genetic variation Structural Deletions Inversions Insertions Repeats VNTRs STRs Single base pair SNPs Point mutations
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DNA Fingerprinting: Using genetic differences to identify individuals
DNA fingerprinting is a set of techniques that analyze variable regions in DNA in order to identify individuals. The “trick” scientists use to spot genetic differences is to focus on regions of DNA that are known to typically vary across individuals. In the lab, we can isolate variable regions of DNA and separate them by size using gel electrophoresis. DNA fragments of different lengths form a unique pattern of bands in gel electrophoresis, or a “DNA fingerprint” that represents an individual’s unique genetic profile.
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RFLPs: Restriction Fragment Length Polymorphisms
RFLPs can reveal multiple forms of genetic variation 1 2 V N T R 1 4 V N T R 2 SNP 3 4 5 3 5 Deletion Restriction site
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Integrated electrophoresis and transilluminator system
Fast Safe Compact Economical
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Gel casting made easy
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Cast 2% agarose gels 5 lanes needed per lab group
Cast 2% gels 0.4 g agarose 20 ml 1X TBE buffer Heat for ~30 sec Add 2 µl GelGreen DNA stain Pour into casting tray with comb
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Micropipetting practice
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Micropipetting practice
Identify volume range Adjust volume Press plunger to FIRST STOP Press plunger to SECOND STOP
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Transfer real liquids: 20, 10, and 5µl
Adjust volume Get a tip Press plunger to FIRST STOP Collect liquid. Release plunger Transfer liquid Press plunger to SECOND STOP Eject tip
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5 “Shark Attack” DNA Samples
Experimental set-up 15 µL 5 “Shark Attack” DNA Samples + 3 µL 6X Gel Loading Dye DNA Ladder Crown Beach Doom Cove Wave Crest Hoppa-Hoppa L C D W H 18 µL
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Load and run the gel 1 Pipette 15 µl of DNA Load carefully into wells
Ready to run! 2 3 Ladder C D W H RUN
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In nature, DNA does not come in tubes Example sources of DNA for RFLP
Virus, phage, plasmid Eukaryotic genome PCR target (amplicon) Short, simple genome Abundant sample Purify and digest DNA Long, complex genome Less abundant sample Purify, digest, and hybridize DNA Specific genetic target Limited starting sample PCR-RFLP
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Example: a complete workflow to study genetic variation
DNA extraction DNA amplification DNA electrophoresis Obtain genetic material… Use PCR to isolate a genetic difference… …Visualize it! Which other techniques can we use?
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Let’s take a peek at our gels
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Visualize results…. Capture images after VIEW 5 minutes 10 minutes
766 bp 500 bp 350 bp 300 bp 250 bp 200 bp 150 bp 100 bp 75 bp 50 bp 25 bp VIEW
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Learn more: www.minipcr.com/dna-dots
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We hope you enjoyed the lab!
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Backup: miniPCR in Forensics STR analysis using capillary electrophoresis
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miniPCR Learning Labs™
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The complete biotech toolkit
Micropipetting PCR Gel electrophoresis Visualization DNA Discovery System™
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