Pre-lab Questions for Lab 1: Isolation of genomic DNA from rockfish (Sebastes)
muscle tissue or equivalent fish species and tissue
Barcoding Life’s Matrix Teacher Manual (Universal Primer Version updated 9/2012)
Directions: Please answer the following questions ON YOUR OWN PAPER and in COMPLETE
SENTENCES. Some of these questions will show up on your exam!
1. What is so special about the mitochondrial DNA? (discuss shape)
2. How many base pairs are found on the mitochondrial DNA?
3. Genomic DNA is abbreviated _________. What is genomic DNA?
4. What kinds of genes are located on the mitochondrial DNA (mtDNA)?
5. What is the first step in getting the gDNA out of the cells? What is the second step?
6. The 1.5 microcentrifuge tubes are to be labeled with what?
7. What should you do with the incubator before you begin?
8. You are to get a pre-labeled tube from teacher with the fish tissue samples. Why is it important to use
the SAME SPECIMEN ID for all tubes in each of the labs in this program?
9. You are going to set a P-200 for what volume? What does the dial in the window look like (draw it)?
10. What do you do if the salts in the digestion buffer have precipitated out?
11. After you add the digestion buffer and changed your tip, what volume of Proteinase K will you be
using? What micropippetor will you use?
12. What is the function of the Proteinase K?
13. What is the function of the digestion buffer?
14. How long will you vortex the tube? What does vortexing do?
15. How long do you incubate the DNA in these reagents?
16. To ensure COMPLETE digestion, what must happen?
17. What do you do with the samples after incubation?
Pre-lab Questions for Lab 2: Purifying total DNA (gDNA) from cell lysates
Barcoding Life’s Matrix Teacher Manual (Universal Primer Version updated 9/2012)
Directions: Please answer the following questions ON YOUR OWN PAPER and in
COMPLETE SENTENCES. Some of these questions will show up on your exam!
1. In the previous lab, what was in the lysis or digestion buffer? What was the purpose of
this buffer?
2. What is the cell lysate? (what’s in it?)
3. How will you get the gDNA/mtDNA from the other molecules in the lysate?
4. What gets stuck to the silica matrix of the spin column? Why?
5. What is the function of the 2 wash steps?
6. What is the function of the water in the last step of the protocol?
7. What is in the collection tube at the end of the lab?
8. Why do you centrifuge the samples for 3 minutes at 12,000 rpm at the beginning of the
lab?
9. What are you doing while the tubes are in the centrifuge?
10. In step 7, you are transferring the supernatant to a clean 1.5 mL tube.
a. What is a supernatant?
b. What is in this supernatant?
c. What volume are you using on your micropipette?
d. BE CAREFUL NOT TO DISTURB THE _________________!
11. What is the role of RNase A in step 9?
12. What is the role of the lysis/binding buffer?
13. What is the role of the ethanol?
14. In step 15, what should you do to make sure you do NOT touch the silica matrix of the
spin column?
15. What should you do if there is still lysate in the spin column? (step 16)
16. EXPLAIN step 17 in your own words.
17. Explain the role of wash buffer 1 and wash buffer 2?
18. What is the importance of step #25?
19. Why do you add water in step #28?
20. Explain step #29 in YOUR OWN WORDS. What is critical in this step?
21. What are you going to be doing when your samples are spinning?
22. What are you going to do when you have transferred your gDNA samples to a freshly
labeled microcentrifuge tube?
Congratulations! Now let’s perform the lab!
Pre-lab Questions for Lab 3: Casting an agarose gel to examine gDNA by agarose gel
electrophoresis Barcoding Life’s Matrix Teacher Manual (Universal Primer Version updated 9/2012)
Directions: Please answer the following questions ON YOUR OWN PAPER and in COMPLETE SENTENCES.
Some of these questions will show up on your exam!
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What is the purpose of an agarose gel?
What type of macromolecule is the agarose?
What buffer will be used in this lab? What is the function of this buffer? (look back in lecture notes
on gels.)
DNA will migrate through the gel in which direction?
Explain how DNA fragments of different sizes migrate through an agarose gel.
How are we going to visualize our DNA in the gel (what compound will be used)?
What piece of equipment will be used to visualize the DNA labeled agarose gel?
What percentage of gel will be used for this lab?
Describe how you will set up the gel (casting) trays prior to pouring your gel. (Read the Fotodyne gel
system)(steps 1-4)
What is the purpose of the labeling or masking tape?
How much agarose will be measured out?
What does the manual suggest you avoid while transferring the agarose powder into your 500 mL
flask?
How much TAE buffer will you use?
Why do you plug the mouth of the flask with paper towels?
Explain how you will microwave your solution.
What does the teacher add to your flask?
Summarize steps 13-16.
Why is there a warning about pouring hot agarose into your gel tray/gel chamber?
Pre-lab Questions for Lab 4: Examining purified gDNA with agarose gel electrophoresis
Barcoding Life’s Matrix Teacher Manual (Universal Primer Version updated 9/2012)
Directions: Please answer the following questions ON YOUR OWN PAPER and in COMPLETE SENTENCES.
Some of these questions will show up on your exam!
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Are you going to load ALL of your DNA samples onto this gel? Explain
What is the indicator dye that is added to the loading buffer?
What is the purpose of this indicator dye?
What other reagent is added to the loading buffer? Why?
If you correctly placed your wells near the negative anode, which direction will the DNA migrate
through the gel?
Which direction will the indicator (loading) dye migrate when the current is applied? Why?
How are we going to visualize the DNA in our gel?
What kind of DNA band should we expect to see (small or large)? Will it be near the wells or far away
from the wells? Why?
For the fotodyne system, it is important to do what to the gates before running your gel?
Where do you want the wells to be when placing the casting tray into the electrophoresis chamber?
Read step #5. After you take your 10 uL of purified gDNA what are you going to do with the “stock”
DNA tube?
How are you going to mix your DNA and loading dye? (step #8)
How are you going to spin your DNA samples?
In your own words, describe step #12.
The ______________________ lead should be located at the bottom of the gel before turning on the power
supply. Why?
What is the voltage and time for our gel?
The dye is going to migrate through the gel at a rate comparable to what?
What are you going to do after the gel is finished running?