In Search of Satellite DNA in Callosobruchus maculatus
Instructor Notes
Question to be addressed: Does Callosobruchus maculatus have satellite DNA?
Notes to Instructors
Describe the pre-laboratory planning you expect students to do and/or describe the decisions that
must be made prior to conducting an experiment. Be as explicit as possible for example you may
present a bulleted list asking students to:






Describe an experimental design for evaluating hypotheses.
Predict the outcomes for the experiment.
Identify and list the variables you would manipulate in the experiment.
Identify and list the variables you would keep constant in the experiment.
List the data you would collect to determine if your predictions were true.
Come to class prepared to present your experimental design.
We provide students with a pre-laboratory handout
(http://www.beanbeetles.org/protocols/satellite_DNA/downloads.html) with reading
assignments, links to tutorials on DNA analyses using restriction enzymes and gel
electrophoresis, and questions on bean beetle life history and molecular biology techniques.
Requiring that students complete the pre-laboratory assignments helps improve their ability to
conduct this study.
Experimental Design
Null hypothesis
There are no repetitive sequences in bean beetles.
Alternative hypothesis
There are repetitive sequences in bean beetles.
What variables should be kept constant in the experiment?
DNA extraction protocol, digestion conditions, gel electrophoresis
What variables may be manipulated?
The restriction enzymes, restriction digestion time, the host bean for the beetle strain, sex of the
beetle, genome from other species
What will be measured, observed, or tabulated in this study?
The DNA banding pattern and sizes of repeated sequences
1
Predictions for the null hypothesis
If there are no repeated sequences (satellite DNA), no DNA banding pattern will emerge (only a
smear on the gel).
Predictions for the alternative hypothesis
If there are repeated sequences (satellite DNA), a DNA banding pattern will emerge. Different
restriction enzymes should yield different patterns, diffuse bands (no clear pattern), or no pattern
(smear).
What are the control treatments in this experiment?
Isolated DNA will be incubated in the presence or absence of at least two different types of
restriction enzymes, with the reactions being allowed different digestion times. Results will be
compared to published data obtained from Tenebrio molitor or other species which has been
shown to have satellite DNA.
What issues should you as the instructor remind students to consider concerning
experimental controls, confounding variables, and replication?
Remind students that satellite DNA is NOT used to differentiate between individuals, but it is
more used as a marker for a species. You can guide your students to ask question like these:
 Are there differences between subpopulations? Just as very dissimilar species have the
same number of chromosomes, it is possible for different species to have same size
satellite DNA.
 What is the DNA sequence of the bean beetle satellite? That question cannot be answered
by this experiment, the satellite DNA must be sequenced to answer that question (This is
an example of the direction in which this research could go – maybe your students will be
able to pursue this question at your institution!)
Data Collection
Due to technical limitations at our institution, I isolated the DNA and provided the DNA to my
students. They decided which enzymes to use, carried out the digestions, and ran the gels.
Data Analysis
Students should calculate the sizes of their bands. There are no alternative means of analyzing
the data.
Equipment and Supplies
What follows is a step-by-step explanation of how DNA was extracted starting with 50 beetles.
That yields enough DNA for 5-6 samples to be digested and loaded on the gel (or not digested,
as a control). That should be sufficient for one working group of students. If you have groups of
3 performing the experiment, for example, each of the 10 groups in a class of 30 students will
need to do the following steps if they are isolating the DNA themselves. Any biotechnology lab
will have the equipment needed for the experiments. If the equipment is not available, the DNA
2
could be extracted somewhere else and provided to the students so they could perform the
digestions and run the gels. In our experiment, we used ethidium bromide gels, but EtBr is toxic,
so after running the electrophoresis of the samples, I handled the gels, photographed them and
then gave the photos to my students. SYBR safe or GEL RED might be considered as
alternatives, as they are safer products.
Laboratory Methods
Day #1 – DNA Extraction
This protocol is briefly presented as a paragraph in the student’s handout without details, but I
am including these steps to make it easier to reproduce the results we obtained. These steps are
described in the protocol included in the Promega DNA Extraction kit A1120 and all the
solutions mentioned are included in the kit. One kit provides enough materials to use for several
semesters.
1. Freeze bean beetles (bb). Select 50 bb and place in 1.5ml microfuge tube.
2. Crush with a glass rod and use a toothpick to get to the beetles in the bottom of the
microfuge tube.
3. Add 900 ul of Cell Lysis Solution. Invert to mix.
4. Incubate for 10 min. at room temp.
5. Centrifuge 20 sec at 13-16k x g
6. Discard supernatant (NOTE: See Sample Results, Figure 1, lane 3 shows that there is no
DNA in the supernatant.)
7. Add 600 ul Nuclei Lysis Solution and 3 ul RNase A
8. Mix and incubate at 37 oC for 15-30 min. After incubating, cool to room temp
9. Add 200 ul Protein Precipitation Solution and vortex
10. Chill on ice for 5 min
11. Centrifuge 4 min at 13-16k x g
12. Transfer supernatant to clean tube that contains 600 ul isopropanol at room temp
13. Mix gently by inversion
14. Spin at 13-16k x g for 1 min
15. Remove and discard supernatant
16. Add 600 ul of room temp 70% ethanol to the pellet. Mix gently by flicking the microfuge
tube
17. Spin at 13-16k x g for 1 min
18. Aspirate ethanol (being careful not to disturb the pellet) and air-dry pellet for 15 min
19. Rehydrate the DNA in 100 ul of DNA Rehydration Solution for 1 hour at 65oC or
overnight at room temp.
20. Extracted DNA that has been resuspended in the Rehydration Solution can be stored
frozen at -20 oC until ready to use.
3
Day #2 – Enzyme digestion and agarose gel electrophoresis
1. Thaw DNA in ice
2. Use 10-16 ul of resuspended DNA to either directly load on the gel or digest.
Digestion protocol
1. 10-16 ul of DNA (add dH2O to bring up the volume of DNA + water to 16 ul; the more
DNA you use the easier it would be to see bands)
2. 2 ul of enzyme
3. 2 ul of enzyme-specific buffer (provided with the enzyme by manufacturer)
4. Digest at 37oC (digest for at least 1 hour – students may manipulate this variable, if
desired. NOTE: Samples in the Figure 2 gel [Sample Results] were digested for 2 hours)
5. Add 2ul of loading dye per sample and load entire sample on minigel (add 2 ul of 1%
ethidium bromide to 40 ml of agarose gel when preparing the gel or add EtBr after
running)
6. Run gel for 30-60 min at 70-100 V
7. Take photo of gel under UV light
This experiment was written by Beatriz Gonzalez and Elizabeth Deimeke, 2014
(www.beanbeetles.org).
4