Transcript Tips - Workshops+SJCOE Workshop Management

Grades 11-12 Biotech
Boot Camp 2015
Kirk Brown
Director of Science & STEM
Integration & Innovation S2I2
San Joaquin County Office of Education
Andrew Hutton
B.S. Neuroscience/ B.A. Psychology
University of California, Santa Cruz
Agenda for Day 1
1.
2.
3.
4.
5.
6.
7.
8.
Introductions
Safety Orientation
Lab Notebook Orientation
Pipetting Review and Practice
Solution Making
1.
Percent Solutions
2.
Molar Solutions
Lunch
Ouchterlony Immuno-diffusion Test
Detecting GMO in Dorito Chips Part 1
Notebooks
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Look at notebooks
Review Rubric
Use as a tool to record
what you do as you do it
Some tips
Pipetting
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Transfer Pipet
Use
Goals:
Transfer 250μl,
500μl, 750μl and
1 ml of colored
water 24 well
plate.
Each partner
will complete
task
How might you
measure the
accuracy?
Micropipetting
Micopipetting Video
• Goals:
1. Pipet a series of
droplets on a piece
of wax paper.
•
2,4,6,8,10,12 μl droplets
in order
2.
3.
4.
Show Instructor
Write your initials in
3 μl droplets
Show instructor
Micropipetting
Goals:
1.
Pipet 45 μl of colored
water into a 1.5 ml
microcentrifuge tube.
2.
Transfer 5 μl into a
new 1.5 ml
microcentrifuge tube
3.
Transfer 15 μl of
colored water from
the 1.5 ml tube into
the 1.5 ml tube that
already has 5 μl in it.
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Making Solutions
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Percent Solutions
Making Solutions
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Molar Solutions
GMO Detection by PCR
• Research
•
question:
Does the food tested contain genetically modified plant material?
• Objectives:
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Grind one test food and one non-GMO control food using a mortar and
pestle
Extract gDNA with InstaGene matrix
Set up six PCR reactions with test food gDNA, gDNA from non-GM
control food and GM-positive plasmid DNA using GMO master mix (red)
and plant master mix (green) for each sample
Load and run PCR products on 3% agarose TAE gel
Determine whether the test food has been genetically modified
Safety Reminders
•
Follow all laboratory safety procedures
Wear appropriate PPE
• Be careful when using 10% bleach, it can
harm clothes and is hazardous
• Remember to balance centrifuge and
ensure inner lid is on
• Be careful with electricity in the presence
of liquids
• If ethidium bromide or SYBR Safe stain are
used, follow appropriate safety precautions
•
Skills to Master
• Use
mortar and pestle
• Perform DNA extractions using
InstaGene matrix
• Set up PCR reactions
• Use a thermal cycler
• Perform agarose gel electrophoresis
• Analyze results on an agarose gel
•Tips
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PCR is very sensitive; watch for contamination of reagents
or tips
Mortar and pestle is a source of contamination and should
be wiped with 10% bleach and rinsed with tap water
followed by distilled water
Too much plant material can overwhelm the InstaGene
matrix. Use only the recommended amount
Use a transfer pipet to transfer the ground food to the
InstaGene matrix since micropipet tips are easily clogged
Use aerosol barrier tips when setting up PCR reactions
Keep master mix and reaction tubes on ice until they are
ready to be placed in thermal cycler
Mix reagents thoroughly and ensure reactions are at bottom
of tubes
Student Workstation Materials
Student Workstation Materials
Protocol Highlights/Tips
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Use the proper amount of plant
material and dilute with 5 ml of water
for every gram of plant material
Use a transfer pipet to transfer plant
material to InstaGene matrix
Use aerosol barrier pipet tips
Keep tubes on ice
Keep a map of tube location in thermal
cycler
Ensure that reagents are mixed well
and on bottom of tubes
Minimize contamination
3% TAE gels should be used to run
samples
Using PCR to Detect GM Sequences
• Play
video: GMO Detection by PCR
•Gel Results
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Compare bands to the PCR MW standard
Compare lanes 1, 3 and 5 to see if plant DNA was amplified
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Since lane 1 and 5 are controls, both should amplify plant DNA
Compare lanes 2, 4 and 6
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Since lane 6 is a positive control, and lane 2 is a negative control, compare the results of lane
4 to both
•Summary
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Make sure to:
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Record all steps in your notebook
Keep tubes on ice and watch for contamination
Record the location of samples in the thermal cycler
Run samples in a 3% gel due to their small length
Compare PCR products to determine if the plant material was
genetically modified or not
Ouchterlony Double
Immunodiffusion Assay
• Research
•
question:
Which of the test samples is from chicken?
• Objectives:
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Prepare the Ouchterlony assay plate
Add the antibody and the test samples to the plate
Interpret the results
Safety Reminders
• Follow
all laboratory safety procedures
Wear appropriate PPE
• Take proper precautions when handling raw
meat juices, especially chicken
• Disinfect all surfaces that come into contact with
raw meat or blood proteins
• Wash hands thoroughly after any contact with
raw meat or blood proteins
• Read and become familiar with all MSDSs for this
activity
•
Skills to Master
 Perform
an Ouchterlony double
immunodiffusion assay
Tips
Be sure to change tips between samples and
reagents to avoid sample cross contamination
• When making the wells, it is helpful to depress
the bulb on the cut transfer pipet before
inserting into the PBS agarose
• Release the bulb slightly and then wait until the
plug comes into the transfer pipet
• Plates can be incubated in a small tub with wet
paper towels agar side down until all of the
sample has absorbed into the agarose
•
Student Workstation Materials
Determining Antibody and Antigen
Interaction
• Play
video: Ouchterlony Assay
Protocol Highlights/Tips
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Cut a transfer pipet on a cutting board
so that the pipet sizes are parallel and
no graduations are in the way
Punch the holes 1 cm from the center
whole as shown in protocol
Change tips between samples to avoid
contamination
It may take two days to clearly show
precipitation lines
Results
• Precipitation
the wells
lines can be seen between
Summary
• Make
sure to:
Record all steps in your notebook
• Make sure the wells are 1 cm from the center
and clearly labeled on the plate
• Place the plates into a tub with a lid that has
moist paper towels in it to present drying
• It may take up to two days for the precipitation
line to show up
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Today’s + and Δ
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What did you learn?
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What did you like about
today?
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What would you like to
change or modify?
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Homework!
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Only if you want to!
Agenda for Day 2
1.
2.
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6.
GMO PCR Gels
Gel Analysis
PCR Background
Lunch
PV-92 Chelex Extraction/PCR Set-up
Bacterial Plate counts and serial dilution
Loading a Gel
• Add
4 μl of 6x Uview Loading Dye to each
sample.
• Mix by pipetting
• Load 20 μl in each well
Lane
Contents
1
Neg Control: PMM
2
Neg Control: GMM
3
Test: PMM
4
Test: GMM
5
Pos Control: PMM
6
Pos Control: GMM
7
PCR MW Standard
Agarose
Electrophoresis
Power Supply
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Electrical current
carries negativelycharged DNA
through gel towards
positive (red)
electrode
Loading a gel
Buffer
Dyes
Agarose gel
•Pouring an Agarose Gel
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Casting a Gel
•Deoxyribonucleic Acid
(DNA)
DNA Diagram
Human Modeling of Nucleotide
Phosphate
Base
Sugar
How PCR Works
• http://www.bio-
rad.com/webroot/web/movies/lse/products
/programs/04-0522_PV92_PCR.html
Detection of the Human PV92
Alu Insertion
• Research
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questions:
What is the genotype for the PV92 Alu insertion on chromosome 16 of
the samples in your team?
What is the frequency of the PV92 Alu element in the class?
• Objectives:
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Collect a cheek cell or hair follicle sample
Extract gDNA with InstaGene matrix
Set up a PCR reaction
Load and run PCR products on a 1% agarose TAE gel
Determine the genotype of DNA samples
Calculate the frequency of PV92 Alu element in the class population
Safety Reminders
•
Follow all laboratory safety procedures
Wear appropriate PPE
• Handle only your own saliva sample and
not others
• Remember to balance centrifuge and
ensure inner lid is on
• Be careful with electricity in the presence
of liquids
• If ethidium bromide or SYBR Safe stain are
used, follow appropriate safety precautions
•
Skills to Master
• Perform
DNA extractions using
InstaGene matrix.
• Set up PCR reactions Use a thermal
cycler
• Perform agarose gel electrophoresis
Analyze an agarose gel
Tips
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When extracting cheek cells, gently chew the inside of mouth to help
loosen epithelial cells while swishing with saline
A small pellet of cells should be visible once the samples have been
centrifuged. If not, add more mouth rinse and repeat centrifugation
When samples are incubated at 56ºC, shake them after 5 minutes. This
ensures that more cells break open at 95ºC
PCR is very sensitive; watch for contamination of reagents or tips
Use aerosol barrier tips when setting up PCR reactions
Keep master mix and reaction tubes on ice until they are ready to be
placed in thermal cycler
Mix reagents thoroughly and ensure reactions are at bottom of tubes
Student Workstation Materials
Student Workstation Materials
Determining Alu PV92 Genotype by PCR
• Play
video: Alu PV92 Detection by PCR
Protocol Highlights/Tips
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Chew cheeks for maximum extraction
Be careful not to pour out pellet
Use aerosol barrier pipet tips
Keep tubes on ice
Keep a map of tube location in thermal
cycler
Ensure that reagents are mixed well
and on bottom of tubes
Minimize contamination
1% TAE gels should be used to run
samples
Estimating Bacterial Numbers
Serial Dilutions
• Direct Measurements of Bacterial numbers
• Plate counts: Perform serial dilutions of a sample
Serial Dilution
10 μl
10 μl
90 μl of
broth in
each tube
10 μl
10 μl
10 μl
Plating the Serial Dilutions
100 μl
100 μl
100 μl
100 μl
100 μl
Determining Bacterial Concentration
• Play
video: Serial Dilution and Plate Counts
Today’s + and Δ
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What did you learn?
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Add two more words to the
word wall…
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What did you like about today?
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What would you like to change
or modify?
Agenda for Day 3
1.
2.
3.
4.
5.
6.
7.
PV-92 Gels
Gel Analysis
Bacterial Quantitation Results
Lunch
Gram Staining
Clean-up
Graduation and Certificates
Student Workstation Materials
Gel Results
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Compare bands in lanes 5–8 to three controls
Always ensure that the bands match the controls and use the EZ load
mass ruler as a secondary control to ensure they are approximately
960 bp for (+) and 660 bp (-)
Calculate the number of CFU/ml
Concentration of Bacteria (CFU/ml) =
• Find a plate that has between 20 and 200
colonies
• If the 1/1,000,000 plate has 32 colonies and 100
µl was plated then the following calculation
would determine the CFU/ml
• First step is to multiply the CFU by dilution
factor:
32 CFU x 1,000,000
Answer in
x =
CFU/ml
Amount
plated
Convert
µl to ml
Gram Staining
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Research questions:
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Are bacteria found in yogurt and E. Coli HB101 bacteria
gram-positive or gram-negative?
What are the size and shape of E. coli HB101 and bacteria
from yogurt?
Objectives:
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Mount bacteria on a microscope slide using aseptic
technique
Perform Gram staining of bacteria
Determine gram status of bacteria
Determine cell shape of bacteria
Determine size of bacteria
Safety Reminders
• Follow
all laboratory safety procedures
Review all MSDSs of the stains used in the
activity
• Gram staining has flammable components;
ensure no open flames or spark hazards are
present when Gram staining
• Use aseptic technique when using bacteria
• Wear appropriate PPE
• Do not eat or drink in the laboratory
•
Skills to Master
Perform Gram staining of bacterial
• Heat fix bacteria to slide
• Observe bacteria using a microscope
• Differentiate gram-positive and gramnegative bacteria
• Identify bacteria from cell shape
• Sketch microscopic details
• Estimate size of cells using a microscope
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Tips
• Wear
gloves to avoid staining fingers
• Have multiple beakers of water to use for
destaining
• Wooden clothes pins can be used to hold
slides to heat fix and stain
• The decolorizer used in Gram staining has a
high percentage of alcohol and no flames
should be present when using
• Wear appropriate PPE
Student Workstation Materials
Gram Staining Bacteria
• Play
video: Gram Staining
Protocol Highlights/Tips
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Remember to use appropriate safety
equipment
Make sure the bacteria are dry on the slide
before heat fixing
Follow aseptic technique at all times
Make sure that slide is completely dry after
Gram staining before observing in the
microscope
Make detailed observations in your notebook
Digital cameras and cell phones can be used
to take pictures of bacteria in microscope
through the eyepiece
Summary
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Make sure to:
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Record all steps in your notebook
Make detailed observations after each
step
If oil immersion lenses are available,
use to view Gram stained bacteria at
1,000x
Bleach or autoclave plates/tubes upon
the completion of the experiment
Clean Up!
Thanks and Graduation!
•
Amy Kennedy
•
Ann Carl
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Bret States
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Jane Steinkamp
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James Mousalimas
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You and your parents
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Next Steps:
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Next Summer….
•
Helper