Name: BIOL&211 Lab 9 BIOL&211: MAJORS CELLULAR Lab 9: Molecular Tools &

Name:
BIOL&211 Lab 9
BIOL&211: MAJORS CELLULAR
Lab 9: Molecular Tools & DNA Fingerprinting (Part 1) – 20pts (including online pre-lab 9 and lab 10)
Learning Outcomes
After completing the lab exercises you should be able to:
Accurately and precisely measure fluid volumes with micropipettes.
Explain how restriction nucleases digest DNA.
Describe the basic steps in DNA fingerprinting process.
Explain the basic principles of DNA gel electrophoresis.
Determine the size of unknown DNA fragments.
Explain how DNA fragment lengths are used for identification of individuals.
In this lab, we will use some of the techniques commonly used in molecular biology and forensics laboratories to gather evidence about criminal suspects. Restriction enzymes are enzymes isolated from bacteria that cut up DNA molecules (they do not denature the DNA, they just cut it into smaller pieces). This week we will use restriction enzymes to digest (cut) a DNA sample retrieved from the scene of a crime. We will also use the same restriction enzymes to digest DNA isolated from several suspects. After digesting, we will separate the digested DNA fragments by electrophoresis (lab 10).
We will use a technique called agarose gel electrophoresis to separate the fragments of DNA by size. Finally, we will measure the migration distance in the gel of the DNA fragments, determine their relative sizes (the DNA sample “fingerprints”), and identify the alleged guilty suspect.
To prepare for Fingerprinting Lab and your pre-lab view the videos below learn more about restriction fragment length polymorphism (RFLP), aka DNA fingerprints.

Great animation; obnoxious sound effects. Click on the link below the animation (“Can DNA Demand a Verdict?” to learn more on using DNA in criminal cases) http://learn.genetics.utah.edu/content/labs/gel/
For another tutorial on gel electrophoresis, view this animation:
http://www.dnalc.org/resources/animations/gelelectrophoresis.html
First we will learn how to use pipettes.
Experiments in cell and molecular biology often require the allocation of very small amounts of liquid. Thus, special instruments called micropipettes (or pipettors) are designed to aliquot these small volumes accurately and precisely. Micropipettes are used to pipette volumes in the range of 1 μL to 1000 μL (or 1 mL). Micropipettes have dial-a-volume designs, where you can dial in the exact volume you wish to pipette. However, each micropipette has limitations! For example, one pipettor is only accurate in the volume range of 20 μL to 200 μL. Pipettes are named by the maximum volume they can aliquot (pipette). The 20-200 μL, then, is called a “P200”.
Depressor
Dial

a

Volume
Disposable tip

In this micropipette, the volume is set to 50 microliter.

The plastic disposable tip on the end is where the
liquid is drawn up and held during the transfer.
Depressor
Dial

a

Volume
Disposable tip

In this micropipette, the volume is set to 50 microliter.

The plastic disposable tip on the end is where the
liquid is drawn up and held during the transfer.
Good technique with a micropipette is essential to accuracy. How can you tell if your pipetting technique is accurate? One way is to do trial runs by weighing water. For example, let’s say that you want to pipette 50 μL of water. It turns out that 1 mL of water weighs almost exactly 1 gram.
Since 1000 μL = 1 mL
50 μL = 0.05 mL
0.05 mL = 0.05 g
Thus, the volume of water you pipette should weigh exactly 0.05 g if your technique was perfect.
Complete the following conversions from μL of water to grams and vice versa.
357 μL = _________ mL = ____________ g
10 μL = _________ mL = _____________ g
57 μL = _________ mL = _____________ g
Joe Labhack pipettes a drop of water that weighs 32 mg. How much liquid did he pipette?
Lab Exercises
A. Micropipettes
Watch the instructor demonstration on micropipettes. Then answer the following questions.
1) How many different micropipettes are available today (measured by their volume ranges)? ______________
What are their labels (i.e. 2 – 20) and what volume range can they accurately aliquot?
Label: ________________ Volume range: __________________ μL
Label: ________________ Volume range: __________________ μL
Label: ________________ Volume range: __________________ μL
Which micropipette will you select if you need to aliquot the following volumes?
15 μL: _____________________ 850 μL: ______________________
300 μL: ______________________ 25 μL: ______________________
142 μL: ______________________
Now select a number between 10 and 1000. Write it here: ____________________ This is volume of colored water (in μL) that you will aliquot.
If your technique is perfect, how much should this volume of water weigh? ___________
Set the micropipette to the correct volume and aliquot out the appropriate amount of colored water: first tare the weigh boat, then add your pipette solution and close the lid.
What is the weight? ___________________ (INCLUDE UNITS!). Does this equal your predicted weight? If not, check with me for technique. Your weight may vary since there’s vibrations and air movement in the lab.
When you feel proficient at pipetting, I will assign you a new volume to aliquot (it’s written on the board).
Instructor assigned volume: _________ μL
Actual weight: _____________________ (INCLUDE UNITS!).
Discuss with your lab partners why you should change disposable tips EACH time you move a solution when handling an authentic biological sample. Also note that the pipette tips are sterile- don’t touch the tips with anything but the pipette, and always close the tip box when you’re not taking a tip!
B. DNA Fingerprinting Lab Procedure, Week 1
Materials
– EcoRI/Pst I enzyme mix (ENZ) – Micropipetters and sterile tips
– Crime Scene DNA
– Working plastic microtube rack
– DNA from suspects 1-5
– Ice in container
– Lab marker pen
– Color-coded sterile microtubes
– 37°C water bath
– Floating Styrofoam microtube rack
– Waste container
On ice, in the center of your bench find microfuge tubes labeled as follows:
Crime scene (CS)
Suspect 1 (S1)
Suspect 2 (S2)
Suspect 3 (S3)
Suspect 4 (S4)
Suspect 5 (S5)
Each tubes contains 10 ul of DNA from the either the crime scene or a suspect. This is a small volume and you may not see it, but it’s there!
Label each of the six microtubes with a symbol your group will recognize later (star, number, squiggle, smiley face, whatever you like….).
Place the tubes in the solid plastic rack provided (the Styrofoam racks are not used when you manipulate the tube contents). Set the micropipetter to a volume of 10 µL and affix a clean tip.
Keep the enzyme solution on ice when you’re not taking out an aliquot! Using a fresh tip for each sample, transfer 10 µL of the restriction enzyme mix (labeled ENZ) from the stock tube into each tube of DNA. Repeat for each of the suspects’ DNA samples. (Note: Each stock sample contains DNA diluted in a buffer solution to maintain the correct pH needed for enzyme activity.)
Close the caps on all the tubes and flick gently 2-3 times to mix.
Centrifuge for 5 seconds- make sure to balance the centrifuge! (Instructor or technician will guide you in this process.)
Incubate the samples at 37°C for around 60 minutes; this time frame will let the digestion reaction go to completion. After the incubation, place your floating racks in the collection tub provided. The tub will be stored until the next lab period (FWS quarters only) OR prepped to run on gels right away (summer quarter).
While your samples are incubating practice loading submerged mock gels after the in-class demonstration. Use 15µl per sample.
Questions
Why is it important in step #4 above to change tips if, during pipetting enzyme into each tube, you touch the tip to your fingers or the DNA sample?
Why might it be OK to touch the tip to the inside wall of the DNA sample tube?
What is happening to the DNA molecule as the reaction mixture incubates at 37°C? Why do we use 37°C for incubation (consider the enzyme source)?
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