PTC Lab Protocol
DAY 1: DNA isolation
1.
2.
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6.
Rinse mouth with salt solution (saline) for 30 seconds and spit back into cup.
Label tube with assigned # and transfer 1,000l from cup to tube.
Spin in balanced microcentrifuge for 90 seconds.
Pour off top liquid (supernatant) keeping pellet.
Pipette up and down to mix it up again (resuspend).
Transfer 30l of cells to tube with 100l Chelex.
7.
Add a boiling cap to tube and boil in heat block at 99C for
10 minutes.
8.
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11.
Shake vigorously for 5 seconds.
Spin in balanced microcentrifuge for 90 seconds.
Label tube with your number and transfer 30l taken from the top (supernatant).
Store in the freezer until DAY 2.
PTC Lab Protocol
DAY 2: DNA amplification by Polymerase Chain Reaction (PCR)
1.
Label a small PCR tube with your #. Add 23l of primer/dye mixture to tube with PCR
bead (white).
2.
3.
Add 3l of your DNA saved from DAY 1 to the PCR tube.
Place tube in small holder with the rest of class and store on ice. Samples should be
placed in thermal cycler within 24 hours.
PTC Lab Protocol
DAY 3: Digestion of PCR Product with HaeIII enzyme
1.
Spin PCR tube (in adapter) for 15 seconds. Transfer 12l of the PCR product
to a new tube and label with your # and D for digested. Save PCR tube for Day 4.
2.
Add 2l HaeIII enzyme to “Digest” tube (NOTE to teachers: HaeIII should first be
diluted 1:1 in 50% glycerol).
3.
Mix gently.
4.
Spin for 5 seconds in a balanced centrifuge.
5.
Incubate at 37C for 30 minutes. Then store in freezer with PCR
tubes.
PTC Lab Protocol
DAY 4: Gel Electrophoresis
1. Plug the Power Base unit (Red) into an electrical outlet. (It will beep and show lights)
2. Take Gel out of foil packet carefully, keeping the comb in place.
3. Insert the gel into the Power Base top (with comb and showing the Invitrogen symbol at bottom) right
edge first. Press firmly at both top and bottom until a steady red light shows at top.
4. Pre-run the gel (with comb still in place) by pressing and holding either button until the red light turns to
a flashing green light, release the button and let gel run for the 2 minute pre-run, it will stop
automatically.
5. Remove comb from gel and load samples as indicated below.
Undigested: Load entire sample (14 ul) from PCR tube containing undigested product.*
Digested: Load entire sample (14 ul) from the “Digested” tube.*
Please note that the DNA marker used is the 100 base pair ladder. Load 10 ul of this ladder.*
Load 20l per well of water in remaining wells.
*IF e-gels containing sybr-safe have expired, you must first add 2 ul of sybr-safe to all samples prior to
loading. After adding sybr-safe, load the entire sample in each lane.
6. Press and release either button to start the gel running.
7. After gel has run, take the gel out of the Power Base and place on blue light box with orange cover to
visualize results.
Undigested
Digested
Undigested
Digested
Undigested
Digested