JACK THE RIPPER MEETS DNA TECHNOLOGY
A DNA ANALYSIS SIMULATION

Developed by: Joyce R. Calo and Deborah Corey
© American Association of Immunologists 1997

Overview
Genetic engineering is an integral part of any biology course whether an introductory or advanced placement class. One can hardly pick up a newspaper, magazine, or book or turn on television without encountering uses of genetic engineering. Students need to understand the basics of this new technology to make educated decisions whether personally in their everyday lives or as part of society.

In this experiment, students will amplify the DNA samples provided using the process of polymerase chain reaction (PCR). These amplified samples will then be loaded into the wells of an agarose gel and undergo electrophoresis. An electrical field applied across the gel causes the negatively charged DNA fragments to move through the agarose gel towards the positive electrode. Smaller fragments will move more quickly than larger pieces. This results in distinct bands being formed.

Once stained, using ethidium bromide or methylene blue, the patterns are made visible. Students will then compare the banding pattern formed of the unknown samples of DNA with bands formed by fragments of known sizes.

In order to have the students perform all of the laboratory activities in this packet, a considerable amount of time must be devoted to the background information, laboratory preparations, the lab activities, and the data gathering and follow-up analysis. Students need to have a solid background of the structure of the DNA molecule. Having performed other separation techniques such as the separation of plant pigments using paper chromatography will provide a reference activity with which the students can associate.

Learning Objectives
After completing this laboratory exercise, the student will be able to:

  1. explain the three steps necessary for the polymerase chain reaction to occur
  2. explain the purpose of PCR
  3. accurately use a micropipettor
  4. set up an agarose gel with wells
  5. load wells in an agarose gel
  6. explain why DNA fragments migrate towards the positive electodes in the process of gel electrophoresis
  7. explain why DNA fragments separate according to size
  8. predict the number of bands and placement of these bands of a given sample DNA fragment labeled with restriction enzyme sites after gel electrophoresis has occurred
  9. determine which of various DNA samples given, is "from" Jack the Ripper

Suggested Time Line
Due to time constraints, my students perform only the polymerase chain reaction and the gel electrophoresis activities. My class time is one 50 minute period per day. I have included the pre-lab activities which I performed in order to obtain the vectors with inserts of varying sizes. The inserts were DNA fragments provided by Dr. John Schreiber's laboratory at Rainbow Babies' and Children's Hospital in Cleveland, Ohio. These pre-lab procedures are included in this activity packet and may be done by those classes with ample time to spend on genetic engineering.

Days Required Activity

1

 Mix TBE buffer, aliquot distilled water, pcr buffer, & mineral oil
Set up starter E. coli plates for transformation

1

 Transform E. coli with vector/insert (incubate 24 hours)

1

 Screen E. coli for transformed colonies (incubate 24 hours)

1

 Mini-prep to insure the inclusion of vector & insert
Run gel
Post lab - stain gel

1

 Pre-lab discussion, practice using micropipettors

1

 PCR - set up tubes
Thermal cycler- RB&C

1

 Pre-lab discussion, practice loading wells in agarose gel

1

 Gel Electrophoresis: pour, load, run
Post lab - stain gel

1

 Results & Discussion

Reagents and Solutions

GTE (Glucose/Tris/EDTA) solution

50 mM glucose
25 mM Tris Cl, pH 8.0
10 mM EDTA
Autoclave and store at 4°C

NaOH/SDS solution

0.2 N NaOH
1% (wt/vol) sodium dodecyl sulfate (SDS)
prepare immediately before use

5M potassium acetate solution, pH 4.8

29.5 ml glacial acetic acid
KOH pellets to pH 4.8
H20 to 100 ml
Store at room temperature (do not autoclave)

TBE Buffer

mix 1 packet with1 liter of dH20

DNA Markers

90 ul dH20
10 ul 1.0 kb ladder

Agarose

.8 g agarose
100 ml TBE buffer

Equipment & Supplies For 6 Stations

Transformation

E. coil
micropipettes (0.5 ul- 10 ul and 100 ul - 1000 ul)
micropipette tips (both sizes)
50mM Calcium Chloride
6 incoulating loops
6 culture tubes
Luria broth
Luria broth agar plates
ampicillin
tetracycline
X-Gal, IPTG
6 glass cell spreaders
1 incubator

Screening

pipette
6 culture tubes
micropipette (2.0 ul - 20 ul)
micropipette tips
incubator

Mini Prep

micropipette (100 ul - 1000 ul)
micropipette tips
microfuge tubes
microfuge tube rack
GTE solution
NaOH/SDS solution
potassium acetate solution
100% ethanol
microcentrifuge

Digest

micropipettes
micropipette tips
RNAase
restriction enzymes
buffer
incubator
1KB DNA ladder (GibcoBRL)

PCR

micropipettes (0.5 ul-10ul, 2ul - 20ul and 20 ul - 200 ul)
micropipette tips (both sizes)
6 microtube rack
6 PCR microfuge tubes
10 ul dNTP mix (Gibco 18427-013 use 1 ul)
dATP
dCTP
dTTP
dGTP
10 ul primer 5' (Novagen T4 Promoter primer #689348 - 1 )
10 ul primer 3' (Novagen U-19mer #69819 - 1 )
10 ul Taq polymerase (Gibco 18038 - 042)
15 ul MgCI2
50 ul PCR buffer
50 uldH20
50 ul mineral oil
6 permanent marker
I thermal cycler

Gel Electrophoresis

micropipettes (0.5ul-10ul and 2ul - 20ul)
micropipette tips (both sizes)
6 microtube rack
microcentrifuge tubes
6 gel box
6 power supply
400 ml agarose (Gibco 15510 - 019)
2 liters TBE buffer (Gibco 15546 - 013)
20 ulloading dye
DNA markers (Gibco 1kb ladder 15615 - 016)
methylene blue
1 roll masking tape
1 microwave or hot plate

General

safety goggles
rubber gloves