PTC Lab Protocol – with questions

PART 1: DNA isolation

  1. Rinse mouth with salt solution (saline) for 30 seconds and spit back into cup.
  2. Why are we doing this?
  1. What is the purpose of the salt solution?

2. Label tube with your initials or assigned number and transfer 1,000l (microliters) of your spit from cup to tube. Be sure to pick the correct micropipette.

3. Spin in balanced microcentrifuge for 90 seconds.

a. State the purpose of the microcentrifuge?

b. Why does the pellet go to the bottom?

4. Pour off top liquid (supernatant) keeping pellet, leaving only about 0.1 ml of the fluid. Be careful not to lose the pellet.

a. What’s in the pellet?

5. Set the micropipette to 30 l and pipette up and down to mix the pellet up again (resuspend). Work carefully to minimize bubbles.

6. Obtain and label (with your initials or assigned number) a tube of 100l Chelex. Transfer 30l of cells to the Chelex tube.

a. What is the role of the Chelex?

b. How many total l are in your new tube?______

c. Convert this number of l into ml:______

7. Add a colorful boiling cap to tube and boil in heat block at 99C for 10 minutes.

a. Why do we boil our solution?

8. Shake vigorously for 5 seconds.

9. Spin in balanced microcentrifuge for 90 seconds.

a. Why do we centrifuge solutions?

10. Label a new clean 1.5 ml tube with your initials or assigned number. Use the correct micropipette and a clean tip to transfer 30l taken from the top (supernatant), avoiding the pellet.

Note: We now have your DNA isolated!!!!

PART 2: Preparing DNA for amplification by Polymerase Chain Reaction (PCR)

1. Obtain and label a 0.2mlPCR tube with your initials or assigned number. Add 23l of primer/dye mixture to tube with PCR bead (white).

2. Add 3l of your DNA saved from PART 1 to the PCR tube.

3. Place tube in small holder with the rest of class and store in refrigerator. Your DNA will be taken to Brown University to be amplified by PCR.

a. Why is it that your DNA is being shipped off to Brown (what is the purpose of PCR)?

PTC Lab Protocol

DAY 3: Digestion of PCR Product with HaeIII enzyme

1. Label a 1.5 ml tube containing HaeIII (2ul) with your initials or assigned number. Teacher note: HaeIII should first be diluted 1:1 in 50% glycerol.

2. Spin PCR product tube (in adapter) for 15 seconds. Transfer 12l of the PCR product to the labeled HaeIII tube. SAVE THE REMAINING PCR PRODUCT (small tube).

Mix the “digest” tube gently.

Spin the “digest” tube for 5 seconds in a balanced centrifuge.

Incubate at 37C for 30 minutes. Then store in freezer with remaining PCR product tubes.

PTC Lab Protocol

DAY 4: Gel Electrophoresis

Prepare gels:

Plug the Power Base unit (Red) into an electrical outlet. (It will beep and show lights)

Take Gel out of foil packet carefully, keeping the comb in place.

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.

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.

Prepare samples for loading into gel:

  1. IF gels containing SYBR-safe have expired, add 2ul of Sybr-safe to each of your tubes (DNA ladder, undigested PCR product and HaeIII-digested product). Change pipette tips between samples!
  2. Load 14 ul of either DNA marker, undigested or digested DNA into the appropriate wells. Change tips between samples!
  3. Load 14 ul of water into any empty lanes.
  4. Run gels for 30 minutes.

After gel has run, take the gel out of the Power Base and place on blue light box with orange cover to visualize results.