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Biotechnology Explorer™ | explorer.bio-rad.com 3 Goal Students receive DNA from bones and through restriction digestion and gel electrophoresis see if they match one of two known missing persons
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Biotechnology Explorer™ | explorer.bio-rad.com1
Biomedical Science Activity 1.3.1 DNA Detectives
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PLTW Biomedical Science
1. Show prep1. Recommendations
1. Further dilute Enzymes so adding 5ul not 1ul
2. Stopping points2. Show procedure
1. Fast Blast2. UView
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Goal
Students receive DNA from bones and through restriction digestion and gel electrophoresis see if they match one of two known missing persons
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Preparation
Day 1 Day 2 Day 3 Day 4
Teacher Prep for Restriction Digestion lab & TAE
Student Lab
Restriction Digestion & Gel Pouring
Student Lab
Gel Electrophoresis
Student Lab
Gel analysis
Order kit up to 1 year out
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Preparation
Order kit 1660030EDU Receive kit and store reagent bag at -20C
If you don’t have -20C freezer and you want to store biologicals long term you can freeze the items in a block of ice, which prevents freeze/thaw cycles and ultimately ice crystals from “knifing” the biological material to bits. A styrofoam cooler in the freezer could also work.
Bio-Rad Guarantees kits for 1 year from date received
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Preparation – “bone sample” DNA digests
Add 220µl of sterile water to Suspect 1vial Add 200µl of sterile water to Suspect 2 vial
Mix and allow to sit for 15 minutes
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Preparation – “bone sample” DNA digests
Reaction1
Reaction2
Sterile Water
40.5 µl
DNA Suspect1
45 µl
HindIII 4.5 µlTotal 90µl
Sterile Water
40.5 µl
DNA Suspect1
45 µl
PstI 4.5 µlTotal 90µl
Reaction 1 Reaction 2
37C, 45 minutes
Or
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Preparation – aliquot Bone DNA digestions
Reaction1 B
one HindIII
10µl
X 8R
eaction2 Bone P
stI
10µl
X 8
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Preparation – Lambda DNA Ladder
100µl Lambda HindIII digest DNA
20µl Sample Loading
*65C, 5 minuteslam
bda
*best done day of gel electrophoresis
Dye
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Preparation – aliquoting reagents (PLTW)
Missing 1
Missing 2
Marker
HindIII
Pst1
Sterile w
aterLoading D
ye
X 8 of each
11µl
11µl
12µl
3µl
3µl
50µl
30µl
Lambda
HindIII
Pst1
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Preparation – aliquoting reagents Damon’s Recommendation
Missing 1
Missing 2
Marker
HindIII
Pst1
Loading Dye
X 8 of each
11µl
11µl
12µl
3µl + 12µl water
30µl
Lambda
HindIII
Pst1 3µl + 12µl water
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Preparation – storage recommendation
Missing 1
Missing 2
Marker
HindIII
Pst1
Sterile w
ater
Bone H
indIII
Bone P
stI
Loading Dye
Add to foam float, since these will all be used 1st lab day to do the digestion of DNA
Keep these for the Gel Electrophoresis day
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Preparation – TAE
Make 1x TAE from 50x TAE
You’ll need ~ 3L of 1x TAE for the lab:– 60mL 50X TAE– 2,940 mL distilled water
– OR to a 1Gallon (3.78L) of Distilled water• Add ~76.5mL 50X TAE
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Preparation – 1 X Fast Blast Stain
Add 1mL of 500x Fast Blast Stain to 500mL of Distilled Water. Drinking water also works.
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Preparation – Lab day 1 workstation (8)
Missing 1
Missing 2
HindIII
Pst1
Sterile w
ater
4 clean microcentrifuge tubes
*
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Bones police recovered and worked by your classmates have helped narrow down the field to two missing persons. You will be using restriction digestion and control samples from the two missing persons to determine whose remnants were found.
Student Lab - Scenario
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Student Lab - Scenario
Missing Person #1- Jack Matthews, 21, a senior at the local university, left his apartment at approximately 10 am headed for his Biotechnology class. When Jack did not show up for class, his lab partner alerted his roommate; when Jack did not show up at the apartment that night, his roommate called the police and Jack’s parents. Three months have passed and no one has heard from Jack. Two witnesses remember seeing him on the path near the park on the day he disappeared, but the police have no other leads.
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Missing Person #2- Robert Jones, 24, a newly married insurance salesman, has been missing for almost six months. On the day he disappeared, he spoke to his wife on the phone at approximately 4pm. He told her that he was going to stop at the gym for a workout before coming home; however, the gym has no record of his signing in that day. His wife admits that they were having trouble adjusting to their new life, but she never thought he would simply up and run away.
Student Lab - Scenario
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Sterile w
ater
Label 4 Microcentrifuge tubes 1-4
In notebook, record what will be in tubes
*Add 4µL sterile water to each tube
Student Lab - Procedure Day1
12
34
Tube 1 – Missing Person #1 cut with HindIII enzymeTube 2 – Missing Person #2 cut with HindIII enzymeTube 3 – Missing Person #1 cut with PstI enzymeTube 4 – Missing Person #2 cut with PstI enzyme
* don’t add if you are using Damon’s recommendation of pre-diluting enzymes during the prep
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Add DNA and then *Enzymes to tubes
Close caps, flick to mix and shake down each tube
Student Lab - Procedure Day1
12
34
Tube 1 – 5 µl Missing Person #1 DNA + 1 µl HindIII enzymeTube 2 – 5 µl Missing Person #2 DNA + 1 µl HindIII enzymeTube 3 – 5 µl Missing Person #1 DNA + 1 µl PstI enzymeTube 4 – 5 µl Missing Person #2 DNA + 1 µl PstI enzyme
* If following Damon’s recommendation of pre-diluting enzyme, pipette 5ul of each enzyme above
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Place tubes in a float and put in waterbath for 30 minutes and 37C
Store at 4C until next lab day
Student Lab - Procedure Day1
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1% gel = 1g agarose + ~99g (mL) 1x TAE Each 7x10cm tray needs ~50mL gel
Tape or bumper your trays and add combs In a glass container with 2x head space combine 1X
TAE and agarose. Shake to mix. Microwave in glass container with 2 x head space
with a napkin over the top for 30 seconds. Pull out, gently shake and repeat until solution is
COMPLETELY clear. Pour into tray until gel is half way up teeth of comb
Student Lab – Pour 1% Agarose gels Day1
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Preparation – Lab day2 Gel electrophoresis
12
34
Loading Dye
Samples
Gel
powersupply Gel box
Marker
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Heat Marker for 2 minutes at 95C
Retrieve DNA digest samples 1-4 from 4C Add 2µl Loading Dye to tubes 1-4, flick to mix
Student Lab – Gel Electrophoresis Day2
Loading Dye
Marker
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Set up your gel boxes– Place gels in box– Cover with 1X TAE
Load your gels, record your loading order in notebook
Run 30 minutes at 125V
Student Lab – Gel Electrophoresis Day2
Lane 1: 10 µl Standard DNA MarkerLane 2: 10 µl DNA from Bone cut with HindIIILane 3: 10 µl Tube 1/ Missing Person #1/HindIIILane 4: 10 µl Tube 2/Missing Person #2/HindIIILane 5: 10 µl DNA from Bone digested with PstILane 6: 10 µl Tube 3/Missing Person #1/PstILane 7: 10 µl Tube 4/Missing Person #2/PstI
Can run 20 minutes at 200V if you use 0.25X TAE running buffer
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Remove gels from trays and place in tupperware container.
Cover with 1x Fast Blast stain and let sit overnight
Student Lab – Gel Electrophoresis Day2
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Pour Fast Blast from gels down the sink Fill tupperware with warm water for 1-2 minutes Place gel on white background and photograph
Student Lab – Document Gel Day3
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Student Lab – Analyze Gel Day3
mar
ker
Bon
e
MP
1
MP
2
Bon
e
MP
1
MP
2
HindIII PstI
23,0009,4006,5004,400
2,3002,000
bases
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Student Lab – Analyze Gel – Got math? Day3
mar
ker
Bon
e
MP
1
MP
2
Bon
e
MP
1
MP
2
HindIII PstI
23,0009,4006,5004,400
2,3002,000
bases
1. Measure distance from well to bands in the marker
2. Plot distance versus basepairs
3. Find a line of best fit
4. Use the equation of a line to solve for the size of the other bands
5. Compare the samples based upon basepairs to solve mystery
base
pairs
distance
base
pairs
distance
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What did we use today?
Bio-Rad’s DNA Detectives
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Things I need to bring– Gel caster– UV Transilluminator– Microplate reader– Tupperware x 8- Green Racks 8