Download Activity #3a - Center for Occupational Research and Development

Activity 3: DNA microarray: Hands-on Laboratory Exercise (Using Micropipette)
In Activities #1 and #2, you learned the scientific basis for how DNA microarray technology
works and how it can be used to illustrate variations in gene expression by examining the gene
expression data from two mythological creatures. Different gene expression results in different
characteristics. It is these unique characteristics that endear these mythological creatures to
their perspective cultures. It is their characteristics that allow the myths to thrive and survive
within a culture. In this hands-on activity you’ll examine the gene expression coral reef to see
what genes might help them to survive.
In this experiment, you will determine the gene or combination of genes that gives the coral its
ability or inability to withstand the stress of a transplant successfully! Your microarray will
produce data regarding the varying levels of gene expression of 6 genes that may be involved in
stress and tolerance. These genes are:
1) p53: involved in the initiation of apoptosis (cellular suicide)
2) actin: cytoskeletal component necessary for cell division
3) tubulin alpha chain: necessary for cell growth and maintenance
4) ATP synthase: involved in energy and metabolism
5) 60S ribosomal protein L26: involved in protein synthesis
6) calcium/calmodulin-dependent protein kinase IV: involved in cell communication
Materials:
6 dropper bottles containing genes to spot onto the microarray
2 bottles of hybridization solutions representing the two samples of coral (normal vs. stressed)
** WARNING** contains 0.4M Sodium Hydroxide (NaOH) a basic solution which can
cause burns. Do not get in eyes, on skin or on clothing; wear protective eye-wear and
gloves when handling.
FIRST AID:
Contact: flush with water immediately
Ingestion: seek medical attention
Inhalation: ventilate with fresh air
10 blank re-useable slides for microarray preparation
Gloves and protective safety goggles
Hot water bath (60-65 degrees)
Micropipette set to 30 µl
Sterile micropipette tips
Laboratory notebook
Activity 3: DNA Microarray for Coral Reef
Page 1 of 3
Contextual Biology Integrated Projects
Created by the Center for Occupational Research and Development
http://www.cordonline.net/HiESTbiology
Safety Precautions:

Remember to always practice safe laboratory practices, including washing your hands
before and after your experiment, no eating or drinking in the lab, and wearing all
appropriate safety gear.

The hybridization solutions contain 0.4M Sodium Hydroxide (NaOH) a basic solution
which can cause burns. Do not get in eyes, on skin, or on clothing. Wear protective eyewear and gloves when handling. Follow the first aid procedures below:
FIRST AID for:
Contact: flush with water immediately
Ingestion: Seek Medical Attention
Inhalation: ventilate with fresh air
Procedures:
Prepare the DNA microarray slide:
You will prepare a DNA microarray by spotting six different gene sequences onto a glass
microscope slide.
1. Using a permanent marker, label the red backgrounds next to the spots 1-6, making two
rows of three. Handle your slide by the edges only to prevent touching the surface of
your slide.
2. Write your initials or group number on the label end.
3. Bring your slide to the water bath where the 6 dropper bottles are incubating between
60-65 degrees.
4. Measure 30 µl of solution #1 onto the #1 labeled numbered spot on the DNA microarray
with a micropipette and sterile tip.
5. Repeat step 4 above with solutions #2- #6.
*Remember to switch tips between samples to avoid cross contamination of the
genes.*
6. Record where each gene has been spotted in your laboratory notebook.
7. Replace the dropper bottle lids.
8. Wait for the spots to harden and dry.
9. Your Microarray has been spotted with the 6 genes of interest!
Activity 3: DNA Microarray for Coral Reef
Page 2 of 3
Contextual Biology Integrated Projects
Created by the Center for Occupational Research and Development
http://www.cordonline.net/HiESTbiology
Hybridization of the labeled cDNAs to the microarray:
mRNA was isolated from normal and stressed p. lobata. cDNAs were reversed transcribed and
labeled. mRNA from the normal coral was labeled with a blue dye and mRNA from the stressed
coral was labeled with a red dye.
1. Carefully drop 2-3 drops of the cDNA hybridization solution from the dropper bottle
provided to you onto each spot on your microarray. DO NOT touch the dropper bottle to
the spots.
2. Watch carefully as color appears indicating hybridization. You may move your slide onto
a sheet of white paper for better analysis.
3. Record your results.
4. Use a paper towel to wipe off the six spots on your slide and rinse in running water.
5. Leave to dry on a clean paper towel for the next group.
What can you hypothesize regarding your data?
Activity 3: DNA Microarray for Coral Reef
Page 3 of 3
Contextual Biology Integrated Projects
Created by the Center for Occupational Research and Development
http://www.cordonline.net/HiESTbiology