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Biotechnology in
the High School
Classroom:
A resource guide
and lab manual
for exploring
biotechnology 44
minutes at a
time
Table of
Contents
1
Classroom setup,
equipment, and
supplies
(pp 5 - 46)
1.1
Preparing media
and buffers
1.1.1 Water
1.1.2
Sterilization:
autoclave or
filter
1.1.3 Adjusting
pH
1.2
Student “working
box” kept at
room temperature
1.2.1
Nuclease-free
H2O
1.2.2 DNA
loading dye (6×)
1.2.3 Buffer EB
(elution buffer)
1.3
Shared resources
for the class
1.3.1
Microtubes,
culture tubes,
and centrifuge
tubes
1.3.2 LB media
1.3.3
Antibiotics
1.3.4 Adding
antibiotics to
media
1.3.5 DNA
ladders
1.3.6 Enzymes
1.3.7 Enzyme
reaction buffers
1.3.8 Buffers
and other
solutions for
DNA preps
1.4
Bacterial
Cultures
1.4.1 Bacterial
strains used in
the class
1.4.2
Maintaining
bacterial stocks
during the year
1.4.3 Preparing
stab cultures
1.4.4 Using a
spectrophotometer
1.4.5 Growing
overnight
cultures
2
Methods;
protocols and
suggestions
(pp 47 - 84)
2.1
Restriction
digestions
2.1.1 Reaction
volume
2.1.2 Sample
digestion
parameters
2.1.3 Pipetting
order and other
recommendations
2.2
DNA gel
electrophoresis
2.2.1 DNA stain:
SYBR SafeTM
2.2.2
Pre-pouring gels
with SYBR SafeTM
for students
2.2.3 Loading
volume
2.2.4 Loading
mass
2.2.5 Agarose
concentration,
voltage, and
time
2.3
Chemically
competent cell
preparation (for
transforming
ligation
products)
2.3.1 What makes
cells
"competent"
2.3.2 Chemically
competent cells
are necessary to
transform
ligation
reactions
2.3.3 Notes
about the
protocol
2.3.4 Protocol
2.4
Chemically
competent cell
transformation
2.5
Rapid colony
transformation
2.6
Using Qiagen
columns:
minipreps, PCR
clean-up, and
gel purification
2.6.1 Notes
about Qiagen
kits
2.7
PCR primer
preparation from
lyophilized
stock
2.8
Measuring DNA
concentrations
and purity with
the NanoDrop
spectrophotometer
2.8.1 More about
absorbance: what
is the NanoDrop
measuring?
2.8.2
Determining
purity of DNA
solution:
OD260/OD280
ratio
2.9
Calculating
volumetric
ratios for a
ligation
reaction
3
Lab practicals
(pp 85
- 168)
3.1
Recombination
cloning of two
antibiotic
resistance genes
3.1.1 Timeline
3.1.2 Overview
3.1.3
Preparation
3.1.4
Step-by-step
3.1.5 Reference
gels for
digestions
3.2
“Mystery
Plasmid”
identification
3.2.1
Preparation
3.2.2
Instruction for
the students
3.2.3 Materials
and resources
for students
3.2.4 Sample
student work
from
presentations
3.3
PCR cloning and
His-tagging a
protein for
purification
3.3.1 Timeline
3.3.2 Overview
of practicals
3.3 through 3.5
3.3.3 The
expression
vector
pET-28a(+)
3.3.4 Primers
for amplifying
the DsRed2 ORF
from pDsRed2
3.3.5 Primers
for amplifying
the ZsGreen ORF
from pZsGreen
3.3.6 PCR
amplification of
the DsRed2 and
ZsGreen ORF
3.3.7
Preparation for
cloning
reactions
3.3.8 Verifying
the PCR products
are being
digested
3.3.9 Ligation
reaction:
cloning PCR
product into
pET-28a(+)
3.3.10 Option 1:
transformation
of the ligation
reaction into
DH5α, colony PCR
to verify
inserts,
miniprep, and
rapid colony
transformation
into BL21(DE3)
3.3.11 Option 2:
transformation
of the ligation
reaction into
BL21(DE3)
3.4
Affinity
purification of
His-tagged
proteins
3.4.1 Timeline
3.4.2
Preparation
3.4.3 Harvesting
and lysing cells
3.4.4 Protein
purification
3.4.5 Storing
protein samples
3.5
Analysis of
purified
proteins by
SDS-polyacrylamide
gel
electrophoresis
3.5.1 Timeline
3.5.2
Preparation of
buffers for
SDS-PAGE
3.5.3 Preparing
samples and
running the
SDS-PAGE
3.5.4 Staining
and destaining
the gel for
analysis
3.6
Phage hunting
3.6.1 Timeline
3.6.2 Overview
3.6.3 Media
3.6.4 Collecting
soil samples and
preparing sample
suspensions
3.6.5
Amplification
3.6.6 Assay for
phage activity
4
Appendix
(pp 169 - 187)
4.1
Antibiotics
4.2
Data and tables
4.2.1 Linear
Range of GENESYS
10 UV/Vis
spectrophotometer
4.2.2 E.coli
DH5α overnight
culture density
by media volume
and tube
4.2.3
Restriction
digestion
variables: DNA
mass, reaction
volume, and time
4.2.4 Gel
electrophoresis
variables: gel
concentration,
volume, voltage,
and time
4.2.5 E.coli
DH5α growth
curve
4.2.6 Linear
range of the
NanoDrop ND-100
spectrophotometer
4.3
Equipment list
and information
4.4
Purchasing
information and
sample annual
consumption
4.5
More classroom
references
4.6
References
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