Biotechnology
Explorer Program
Serious About Science Education
Genes in a Bottle Kit :
Capture Your Unique Essence!
166-2300EDU
• Kirk Brown, Tracy High School, Tracy, CA
• Stan Hitomi, Edward Teller Education Center,
University of California, CA
Genes in a Bottle
Workshop Timeline

Introduction

Background on DNA extraction

Extract genomic DNA from cheek cells

Prepare DNA necklaces

Review extension experiments
Why teach DNA Extraction?

Powerful teaching tool
Real-world connections

Link to careers and industry

Tangible results

Laboratory extensions

Why use Bio-Rad’s Genes in a Bottle Kit?
• Provides enough reagents for 36 student DNA extractions
• The activity fits into a single 50-minute period
• Curriculum Manual is set up into two sections:
Basic level instruction (for 5-8 grade)
Advanced level instruction (for 9-14 grade)
• This activity does not require any specialized equipment
• The product is accompanied by Bio-Rad’s world-class
technical support
Genes in a Bottle kit is Suitable for :
• Middle Schools (grades 5-8)
• Secondary/High Schools (grades 9-12)
• 2-year and 4-year Colleges
What can you do with the
Genes in a Bottle Kit ?
• Reinforce the structures of a cell
• DNA structure and function
• Enzyme function
• Introduce students to procedures involved in DNA
extraction
• Preserve student DNA samples in DNA necklaces
• Electrophorese DNA samples on an agarose gel as an
extension experiment
Cell Bio 101

What are the structures of the cell?
Protocol Highlights: Genomic DNA Extraction
 Use 2 soft bristled brushes to collect cells from
the inside of each cheek, and the space between
lips and gums.
 Add cells to lysis buffer to break open cell and
nuclear membranes and to release nuclear
contents.
 Digest sample with protease to degrade proteins.
 Precipitate DNA with cold alcohol in high salt
environment.
Activity
Flow chart
DNA Extraction and Precipitation Workstation set up
4 students/workstation for a total 36 students
Teacher's (Common) Station
Water bath at 50oC
Ice-cold bottle of 91% isopropanol or 95% ethanol on ice
Students’ Workstation (4 students per station)
Number required
Clear micro test tubes, each containing 1 ml lysis buffer
Blue micro test tube labeled “prot”, containing 250 ul of pre-diluted protease
Pink micro test tube labeled “salt”, containing 250 ul of sodium chloride
Clear, capless screw cap tubes
Assorted colored screw caps
Cytology brushes
5 ml round-bottom test tubes
Parafilm (small pre-cut pieces)
Disposable plastic transfer pipets
Foam micro test tube holder
Permanent marker
Disposable paper cup or beaker for waste collection
4
1
1
1
4
8
4
4
4
1
1
1
Ample cell collection is very critical for success.
For best results, make sure to spend the
recommended amount of time collecting and carefully
transferring the cells into the tube of lysis buffer.
Laboratory Protocol
1. Label one clear microtube containing Lysis buffer
(labeled “lysis”) with your initials.
2. Roll the first brush inside your left cheek for 1 minute
and continue scrapping between your cheek and gum and
the roof of the mouth.
3. Place the brush into tube of Lysis buffer and rotate
the brush vigorously to release cells.
4. Scrape on top of tube to get remaining cells off. Put
the brush in a waste container.
Laboratory Protocol
5. Obtain the second brush. Roll the brush inside your
right cheek for 1 minute and continue scrapping between
your cheek and gum and the roof of the mouth.
6. Place the brush into your tube of Lysis buffer and
rotate the brush vigorously to release cells.
7. Scrape on top of tube to get remaining cells off. Put
the brush in a waste container.
Laboratory Protocol
8. Cap microtube containing cheek cells and buffer,
and gently invert 5 times to mix.
9. Add 1 drop of Protease (labeled “prot”) to your
sample.
10. Gently invert 5 times to mix.
11. Incubate sample in a 50C water bath for 10
minutes.
Laboratory Protocol
12. Using a clean plastic transfer pipet, add 2
drops of salt to your microtube .
13. Gently invert microtube 5 times to mix
14. Transfer (pour) your cell extract into a 5 ml
tube.
Laboratory Protocol
15. Slowly add a pipetful of cold alcohol, holding
the tube a 45 angle.
16. Let stand undisturbed for 5 minutes at room
temperature. What do you see?
17. Cover 5 ml tube with parafilm, and gently
invert 10 times to bring DNA out of solution.
Why perform each step?
1. Cell collection
Two soft-bristled brushes are used to dislodge epithelial
cells lining the mouth.
Ample cell collection is very critical for success.
2. Lysis buffer
CH3
CH2
CH2
CH2
CH2
• What is in the Lysis Buffer?
• 50 mM Tris-HCl, pH 8.0
• 1% SDS
CH2
CH2
CH2
CH2
CH2
S
-
O
O
O
– 1% SDS to break open the cell and
nuclear membranes, allowing the DNA to
be released into the solution
– SDS also functions to denature and
unfold proteins, making them more
susceptible to protease cleavage
CH2
O
– Tris buffer to maintain the pH of the
solution at a level where DNA is stable
CH2
SDS
3.Why add Protease?
• Protease is added to destroy nuclear proteins
that bind DNA and cytoplasmic enzymes that
breakdown and destroy DNA.
• Protease treatment increases the amount of
intact DNA that is extracted.
4. Adding salt (5M NaCl)
• Na+ ions of NaCl bind to the phosphate groups of DNA
molecules, neutralizing the electric charge of the DNA
molecules.
• The addition of NaCl allows the DNA molecules to come
together instead of repelling each other, thus making it
easier for DNA to precipitate out of solution when alcohol
is added.
O
Na+
O
Na+
P
O
Base
O
O
CH2
Sugar
O
Na+
Na+
O
O
P
Base
O
CH2
O
Sugar
OH
5. Adding ice cold alcohol?
• DNA cannot dissolve in alcohol
• The addition of cold alcohol makes the DNA clump
together and precipitate out of solution
• Precipitated DNA molecules appear as long pieces of
fluffy, stringy, web-like strands
• Microscopic oxygen bubbles “aggregate” or “fuse”
together, simultaneously with the DNA precipitation
• The larger, visible air bubbles act to “lift” the DNA out
of solution, from the aqueous into the organic phase
Preserving DNA sample:
DNA necklace preparation
1. Using a plastic transfer pipet, carefully
transfer the fluffy DNA strands you
extracted into the small glass vial
-- Transfer as much of your DNA and as little
alcohol as possible
-- The vial should be filled no higher than ½ cm
from the top of the neck of the vial
DNA necklace preparation
continued…
2. Firmly push the plastic stopper cap into the neck of the
vial to seal the glass vial
3. Slip the waxed cord through the silver cap
4. Apply a small drop of Super glue to into the inside of the
silver cap.
DNA necklace preparation
continued…
5. Place the silver cap onto the top of the glass vial and
press down firmly for 30 seconds. Allow the glue to dry
for 10-15 minutes and then check for complete seal
6. After the glue has dried, tie the waxed cord.
Congratulations!
You have just created your very own
DNA Necklace !
How long does the DNA in the necklace last?
The DNA in the glass vial can last for years. Add more alcohol
into the vial if some evaporation occurs.
Genes in a Bottle (166-2300EDU) Kit Components
Kit Provides Enough Materials for 36 Students
 Contains one DNA Extraction Module (166-2000EDU)
two DNA Necklace Modules (166-2200EDU)
DNA Extraction Module (166-2000EDU):
DNA Necklace Module (166-2200EDU):
Protease, 1.3 ml
5 M sodium chloride (salt), 5 ml
Sterile water, 2.5 ml
5 ml round bottom tubes with no lid, 50
Clear, flip-top microtubes, 30
Multicolor, flip-top microtubes, 60
Clear, capless screwcap tubes with assorted color caps, 40
Disposable plastic transfer pipets, 50
Foam microtube holders, 10
Cytology brushes, 80
Parafilm, 1 strip
Curriculum, including a teacher’s guide, and a graphic quick
guide, and separate student instructions for basic and
advanced-level instruction
Glass vials, 18
Silver caps, 18
Plastic plugs, 18
Waxed string, 18
Super glue gel, 1 tube
Instruction manual
(contains enough material for 36 students)
Lysis buffer, 40 ml
(contains enough material for 18 DNA necklaces;
order 2 modules for a classroom of 36 students)
Required Accessories Not Included in Kit:
91% isopropanol (available from drugstores) or 95%
ethanol, 250 ml
Container of ice, 1
Permanent marker, 1–8
Recommended (Optional) Accessories:
Water bath with thermometer 166-0504EDU