It’s as Easy as Pulling a Hair – Student Protocol
Laboratory – It’s as Easy as Pulling a Hair
Student Version
Introduction
The goal of the lab is to isolate DNA from your own hair, amplify a small region of that DNA,
and observe the nucleotide sequence variations of that region by restriction fragment length
polymorphism (RFLP) analysis. Because there are four possible sequence variations in the
amplified region, four different RFLP patterns may be visible on the agarose gel.
Procedures
Part 1: Isolation of DNA from Hair
WEAR GLOVES TO MINIMIZE CONTAMINATION
REMINDER: AS NEEDED, SPIN DOWN TUBE CONTENTS BY PULSING IN A MICROCENTRIFUGE
1. Each student obtains a 1.5 ml microfuge tube containing 400l master mix #1. Label the
tube with your initials.
2. Pluck a single hair—or have your partner help you. Make sure the hair has a root and DO
NOT touch the root, even with gloved hands.
3. Holding the hair above the microfuge tube labeled with your initials, cut ~1 cm from the root
end and let it drop into the tube. IMPORTANT!!! MAKE SURE THAT THE ROOT IS
SUBMERGED IN THE MASTER MIX!
4. Close the lid and incubate at 65oC for one hour.
5. One designated student should obtain a second microfuge tube containing 400l master mix
#1 to set up one control for the class. Label this tube with a “c.”
6. Do not put any hair into the control tube.
7. Incubate the control tube (65oC for one hour) along with the student sample tubes.
8. After the incubation period, poke a hole in the tube tops and boil the samples - including the
control - in a 100oC water bath for ten minutes. This step is necessary to inactivate the
proteinase K.
9. At this point, samples are called “hair DNA” and “control DNA” and can be stored at 4oC
until ready for part 2.
1
It’s as Easy as Pulling a Hair – Student Protocol
Part 2: PCR Amplification of DNA from Hair
WEAR GLOVES TO MINIMIZE CONTAMINATION
REMINDER: AS NEEDED, SPIN DOWN TUBE CONTENTS BY PULSING IN A MICROCENTRIFUGE
1. Each student obtains a 0.2 ml PCR tube containing 50l master mix #2. Label the tube with
your initials.
2. Keep the tube on ice and add 50l “hair DNA” (prepared in part 1).
3. The student preparing the control tube should obtain a second PCR tube containing 50l
master mix #2. To this tube, add 50l of “control DNA” (prepared in part 1).
 Store all left-over DNA samples at -20oC until the experiment is finished.
4. Close the lid and keep all PCR tubes on ice until all groups are ready.
5. Place the PCR tubes in the thermal cycler. Set the program cycle:
94oC for 30 seconds
50oC for 30 seconds
72oC for 1 minute
repeat 30 times
6. At this point, samples are called “hair PCR product” and “control PCR product” and should
be stored in the thermal cycler at 4oC until the next lab period. Samples can then be moved
into a refrigerator at 4oC until ready for part 3.
Part 3: Restriction Digest of PCR Product
WEAR GLOVES TO MINIMIZE CONTAMINATION
REMINDER: AS NEEDED, SPIN DOWN TUBE CONTENTS BY PULSING IN A MICROCENTRIFUGE
1. Each student obtains a 1.5 ml microfuge tube containing 15l master mix #3. Label the tube
with your initials.
2. Keep the tube on ice and add 5l “hair PCR product” (prepared in part 2).
3. The student preparing the control tube should obtain a second microfuge tube containing
15l master mix #3. To this tube, add 5l of “control PCR product” (prepared in part 2).
 Store the left-over PCR product samples at -20oC until the experiment is finished.
4. Close the lid and incubate all tubes at 37oC for 60 minutes.
5. At this point, samples are called “hair digest” and “control digest” and can be stored at 4oC
until ready for part 4.
2
It’s as Easy as Pulling a Hair – Student Protocol
Part 4: Agarose Gel Electrophoresis of PCR Product and Restriction Digests
1. Prepare a 2% agarose gel by mixing 500 mg agarose with 25 ml 1x TBE. One gel is enough
for five student samples (or four student samples and the control sample).
2. Microwave on high for ~20 seconds, or until agarose is dissolved.
3. Pour agarose into a gel casting tray that has been prepared as per instructor demonstration.
4. Before the agarose has solidified, place the 12-well comb into the casting tray.
While the agarose is solidifying, prepare samples for loading onto gel as follows: BE SURE
TO CHANGE PIPET TIPS AFTER EVERY ADDITION!
Tube
Add
hair digest
5l DNA sample buffer (DNA-SB)
new 1.5 ml tube
15l hair PCR product + 5 l DNA-SB
control digest
5l DNA-SB
new 1.5 ml tube
15µl control PCR product + 5l DNA-SB
(only one student needs to prepare the controls; they should go on one gel only)
5. Designate one student (or the instructor) to prepare the DNA molecular ruler. For each gel,
mix 2.5l AmpliSize and 7.5l DNA-SB.
6. Load 10 l of each sample per well as follows: BE SURE TO CHANGE PIPET TIPS
AFTER EVERY SAMPLE!
Lane
Sample
1
2
3
4
5
6
7
8
9
10
11
12
student 1 hair PCR product
student 1 hair digest
student 2 hair PCR product
student 2 hair digest
student 3 hair PCR product
student 3 hair digest
DNA molecular ruler
student 4 hair PCR product
student 4 hair digest
student 5 hair PCR product
student 5 hair digest
blank
7. Run gel at 150mV until first dye front approximately 1 cm from the bottom of the gel. This
should take 40-50 minutes.
3
It’s as Easy as Pulling a Hair – Student Protocol
8. Stain gel and visualize bands.
a) If using ethidium bromide, place gel in ~60 ml 1.0 g/ml EtBr solution. Allow gel to
stain for 10-15 minutes. Pour the EtBr stain back into the bottle and add ~60 ml
water to the gel. Destain in water for 10-15 minutes. Visualize on a uv
transilluminator. PLEASE REFER TO THE APPENDIX FOR CAUTION ON
HANDLING AND DISPOSING OF ETBR!
b) If using DNA safe stain, place gel in ~60 ml stain and cover with plastic wrap. Let sit
overnight at room temperature. Save stain by pouring back into bottle and add ~60
ml water to the gel. Destain in water for ~15 minutes. DNA fragments should be
visible, but it may help to place the gel on a light box or white paper.
4