biotechnology explorer | 1 biomedical science activity 1.3.1 dna detectives

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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


Goal

Students receive DNA from bones and through restriction digestion and gel electrophoresis see if they match one of two known missing persons


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


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


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


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


Preparation – aliquot Bone DNA digestions

Reaction1 B

one HindIII

10µl

X 8R

eaction2 Bone P

stI

10µl

X 8


Preparation – Lambda DNA Ladder

100µl Lambda HindIII digest DNA

20µl Sample Loading

*65C, 5 minuteslam

bda

*best done day of gel electrophoresis

Dye


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


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


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


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


Preparation – 1 X Fast Blast Stain

Add 1mL of 500x Fast Blast Stain to 500mL of Distilled Water. Drinking water also works.


Preparation – Lab day 1 workstation (8)

Missing 1

Missing 2

HindIII

Pst1

Sterile w

ater

4 clean microcentrifuge tubes

*


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



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.


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.



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


Add DNA and then *Enzymes to tubes

Close caps, flick to mix and shake down each tube


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


Place tubes in a float and put in waterbath for 30 minutes and 37C

Store at 4C until next lab day



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


Preparation – Lab day2 Gel electrophoresis

12

34

Loading Dye

Samples

Gel

powersupply Gel box

Marker


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


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


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


Remove gels from trays and place in tupperware container.

Cover with 1x Fast Blast stain and let sit overnight



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


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


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


What did we use today?

Bio-Rad’s DNA Detectives


Things I need to bring– Gel caster– UV Transilluminator– Microplate reader– Tupperware x 8- Green Racks 8

biotechnology explorer | 1 biomedical science activity 1.3.1 dna detectives

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