DNA Detectives Bio-Rad Biotechnology Explorer DNA Fingerprinting Kit
DNA Detectives Bio-Rad Biotechnology Explorer DNA Fingerprinting Kit Crime Scene Have fun setting up your own crime scene. Be as elaborate or as simple as you wish. 2 Biotechnology Explorer | explorer.bio-rad.com Dye Electrophoresis Could you eliminate any suspects using dye electrophoresis? What other methods might be more
conclusive? 3 Biotechnology Explorer | explorer.bio-rad.com Innocence Project 302 DNA exonerations in the U.S. since 1989 (48 in TX) Exonerees served an average of 13.6 years in prison Flawed eyewitness
testimony to blame for many cases 4 Biotechnology Explorer | explorer.bio-rad.com Innocence Project - Resources Innocence Project: www.innocenceproject.org Innocence Project of Texas: www.ipoftexas.org Houston Chronicle profiles: www.chron.com/exonerees 5
Biotechnology Explorer | explorer.bio-rad.com DNA Fingerprinting Real World Applications Crime scene Human relatedness Paternity Animal relatedness Anthropology studies Disease-causing organisms Food identification Human remains Monitoring transplants 6
Biotechnology Explorer | explorer.bio-rad.com DNA Fingerprinting Lab Day 1 7 Biotechnology Explorer | explorer.bio-rad.com DNA Fingerprinting Lab Day 2 8 Biotechnology Explorer | explorer.bio-rad.com DNA Fingerprinting Lab Day 3 9
Biotechnology Explorer | explorer.bio-rad.com How to use a micropipet Play video demonstration or demonstrate live http://www.bio-rad.com/webroot/web/html/lse/support/tutorial_micropipet_wndw.html 10 Biotechnology Explorer | explorer.bio-rad.com Lets Get Started! 1. Place your crime scene (CS) and suspect DNA (S1-5) in your foam rack.
Write your initials on your tubes. 10 ul ENZ 2. Pipet 10 ul of enzyme (ENZ) into each of your tubes. Use a separate tip for each sample! 11 Biotechnology Explorer | explorer.bio-rad.com Lets Get Started!
3. Cap the tubes, flick the bottom of each one to mix, and then bring contents to bottom by tapping on the table. 4. Place your tubes (in the foam rack) in a 37 degree water bath. 12 Biotechnology Explorer | explorer.bio-rad.com DNA Structure 13
Biotechnology Explorer | explorer.bio-rad.com DNA Schematic 14 Biotechnology Explorer | explorer.bio-rad.com Student DNA Model 15 Biotechnology Explorer | explorer.bio-rad.com Restriction Enzymes Evolved by bacteria to protect against viral
DNA infection Endonucleases = cleave within DNA strands Over 3,000 known enzymes 16 Biotechnology Explorer | explorer.bio-rad.com DNA Digestion Reaction Restriction Buffer provides optimal conditions NaCI provides the correct ionic strength Tris-HCI provides the proper pH Mg2+ is an enzyme co-factor
17 Biotechnology Explorer | explorer.bio-rad.com Enzyme Site Recognition Restriction site Each enzyme digests (cuts) DNA at a specific sequence = restriction site Palindrome Enzymes recognize 4- or 6base pair,
palindromic sequences (eg GAATTC) Fragment 1 18 Biotechnology Explorer | explorer.bio-rad.com Fragment 2 5 Prime Overhang Enzyme cuts 19 Biotechnology Explorer | explorer.bio-rad.com
Common Restriction Enzymes EcoRI Eschericha coli 5 prime overhang Pstl Providencia stuartii 3 prime overhang 20 Biotechnology Explorer | explorer.bio-rad.com Classroom Obstacle Course
21 Biotechnology Explorer | explorer.bio-rad.com Restriction Fragment Length Polymorphism RFLP Allele 1 PstI EcoRI CTGCAG GAGCTC GAATTC GTTAAC
1 Allele 2 2 CGGCAG GCGCTC Different Base Pairs No restriction site GAATTC GTTAAC Electrophoresis of
restriction fragments M: Marker A-1: Allele 1 Fragments A-2: Allele 2 Fragments Biotechnology Explorer | explorer.bio-rad.com 3 Fragment 1+2 M 22 3
+ A-1 A-2 How to load an agarose gel Play video demonstration or demonstrate live http://www.bio-rad.com/webroot/web/html/lse/support/tutorial-agarose-gel-electrophoresis-wndw.html 23 Biotechnology Explorer | explorer.bio-rad.com Gel Electrophoresis 1. Collect your DNA
samples from the water bath. 2. Add 4 ul of Uview loading dye (LD) into each of your tubes. Use a separate tip for each sample! Cap the tubes and mix by flicking with your finger. 24 Biotechnology Explorer | explorer.bio-rad.com Gel Electrophoresis 3. Place an agarose gel in the gel box. Make sure the wells are near
the black (-) electrode. 4. Using a separate tip for each sample, load your gel: Lane 1: M, DNA size marker, 10 ll Lane 2: CS, green, 20 ll Lane 3: S1, blue, 20 ll Lane 4: S2, orange, 20 ll Lane 5: S3, violet, 20 ll Lane 6: S4, red, 20 ll Lane 7: S5, yellow, 20 ll 25 Biotechnology Explorer | explorer.bio-rad.com Gel Electrophoresis 5. Place the lid on the gel box, and
plug the electrodes into the power supply. Electrophoresis at 200V for 20 minutes. 26 Biotechnology Explorer | explorer.bio-rad.com Student Inquiry Question to Consider How important is each step in the lab protocol? What part of the protocol can I manipulate to see a change in the results? Possible variables:
enzyme concentration substrate concentration incubation temp or time enzyme or DNA UV exposure methylated plasmid agarose concentration buffer concentration running time. How do I insure the changes I make is what actually affects the outcome (importance of controls). 27
Explorer | explorer.bio-rad.com Biotechnology Write the protocol. After approval do it! Student Inquiry Advanced Question What can I learn about these plasmids? Can I use these plasmids to successfully transform bacteria? Can I ligate these plasmids together and successfully transform bacteria? Can I do a restriction digest on pGLO plasmid? Can I determine the plasmid map using different enzymes? 28 Biotechnology Explorer | explorer.bio-rad.com
Student Inquiry Teacher Considerations What materials and equipment do I have on hand, and what will I need to order? Extra agarose, DNA, different / more restriction enzymes? Water bath (different temps) Other supplies depending on student questions (mini prep, thermal cyclers, etc) Consider buying extras in bulk or as refills many have 1 year + shelf life. What additional prep work will I need? Order supplies Pour gels How much time do I want to allow? Limited time? Have students read lab and come up with inquiry questions and protocol before they start. Collaborative approach. Will you need multiple lab periods?
Will everyone need the same amount of time? 29 Biotechnology Explorer | explorer.bio-rad.com Plasmid Map and Restriction Sites 863bp 863bp 3469bp 2027bp BamHI Hind III EcoRI EcoRI+ HindIII
721bp 721bp 30 Biotechnology Explorer | 947bp 7367bp 1659bp 2027bp 6504bp BamHI: 1 linear fragment; 7367bp
EcoRI: 2 fragments; 863bp / 6504bp 3 fragments; 721bp/2027bp/3469bp HindIII: EcoRI+Hind III: 5 fragments; explorer.bio-rad.com 721bp/863bp/947bp/1659bp/2027bp Electrical current carries negativelycharged DNA through gel towards positive (red) electrode Buffer Dyes
Agarose gel 31 Biotechnology Explorer | explorer.bio-rad.com Power Supply Agarose Electrophoresis Agarose gel separates DNA fragments according to size Electrical current carries (-) charged DNA through gel to (+) electrode.
Small fragments move faster than large fragments Buffer DNA & Loading Dye Agarose gel Power Supply 32 Biotechnology Explorer | explorer.bio-rad.com
Analysis of Stained Gel Determine restriction fragment sizes Create standard curve using DNA marker Measure distance traveled by restriction fragments Determine size of DNA fragments Identify the related samples 33 Biotechnology Explorer | explorer.bio-rad.com Molecular Weight Determination Fingerprinting Standard Curve: Semi-log Distance (mm) 23,000
11.0 9,400 13.0 6,500 15.0 4,400 18.0 2,300
23.0 2,000 24.0 100,000 10,000 Size, base pairs Size (bp) B 1,000
100 0 5 10 15 Distance, mm 34 Biotechnology Explorer | explorer.bio-rad.com 20
A 25 30 DNA Digestion Temperature Why incubate at 37C? Body temperature is optimal for these and most other enzymes What happens if the temperature is too hot or cool? Too hot = enzyme may be denatured (killed) Too cool = enzyme activity lowered, requiring longer digestion time
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