20.109(F16):Asses DNA repair variability with comet chip (Day4)

Introduction

Protocols

Part 1: Load cells and induce DNA damage

For this experiment everyone will use H₂O₂ to induce DNA damage in three Coriell cell lines to assess the DNA repair capacity variability in healthy individuals. Use the following plate map to as you complete the below steps:

Plate map for comet chip repair assay. The labels on the right refer to the Coriell cell lines and the labels on the bottom refer to length of time allotted for recovery following exposure to the H₂O₂. Grey wells will be empty for technical reasons.

1. Load the comet chip prepared by the teaching faculty using the procedure from M1D2; however, please note the following differences:
   • You will not complete the lysis incubation immediately following the cell loading, rather this will be done after the DNA damage induction step.
   • For the cell number you will use 500 K/mL and for the loading time you will use 30 min.
2. While the LMP agar is solidifying at 4 °C, calculate the dilutions of H₂O₂ that you will use for your experiment.
   • The H₂O₂ stock solution is at a ~10 M concentration.
   • You will add 100 μL of 50 μM H₂O₂ per well to induce DNA damage.
3. Retrieve your comet chip from the 4 °C and carefully replace the bottomless 96-well plate to recreate the wells that you used for cell loading.
   • Secure the bottomless 96-well plate with binder clips as before.
4. Obtain an aliquot of H₂O₂ and prepare the concentration that you calculated in Step #2 using 1x PBS as the diluent in a divided [cant remember name of dish] as discussed in prelab.
   • Be sure to keep the H₂O₂ on ice at all times!
5. Add the diluted H₂O₂ to each well of your comet chip that contains cells.
6. Carefully transport your comet chip to the 4 °C cooler and incubate for exactly 20 min.
7. Retrieve your comet chip from the 4 °C cooler and quickly remove the bottomless 96-well plate.
8. Rinse your comet chip using 1x PBS as done during the cell loading procedure.
   • Complete a total of 3 washes.

Part 2: Use comet chip to assess repair capacity

1. Obtain 2 dishes, an aliquot of alkaline lysis buffer, and an aliquot of pre-warmed media from the front laboratory bench.
   • Dispense the lysis buffer and media each into one of the dishes.
2. Carefully cut your comet chip into strips wherein the 6 wells with the same recovery time remain together.
   • Cut through the center of the empty wells to ensure you do not disrupt the experimental wells.
3. Place the 'No drug' and 'O min' strips directly into the alkaline lysis solution.
4. Place the remaining strips into the media and carefully move the dish to the 37 °C incubator.
   • Be sure to set a timer!
5. After 20 min, retrieve your dish from the 37 °C incubator and remove the '20 min' strip.
   • Add the '20 min' strip to the lysis solution.
6. Complete Step #5 after 40 min and 60 min, transferring the appropriate strip to the lysis solution.
7. Move the dish with your comet chip strips to the 4 °C cooler and incubate for 1 hr [have we checked that this is enough time].
8. Remove your comet chip strips from the lysis solution and use a kimwipe to dry the gelbond side.
9. Carefully transport your comet chip strips to the gel electrophoresis station in the 4 °C cold room.
   • The teaching faculty will escort you.
10. Place your comet chip strips on the raised center region of an electrophoresis box.
    • Double-sided tape was applied to the gel electrophoresis box. Be sure you lay your comet chip strips on the tape strips and lightly press down to ensure it is secure.
11. Add enough of the alkaline electrophoresis buffer to the gel electrophoresis box to cover your comet chip strips.
12. Your comet chip strips will incubate in the electrophoresis buffer for 40 min to promote unwinding of the DNA.
13. To separate the damaged DNA into 'comets' it is important that the electrophoresis occur at 300 mA. To maintain the appropriate current, the volume of electrophoresis buffer may need to be adjusted. The teaching faculty will assist you in adding/removing electrophoresis buffer such that this value is reached.
14. The electrophoresis will continue for 30 min.
15. Carefully remove your comet chip strips from the electrophoresis box and place it in a dish.
16. Obtain an aliquot of neutralization buffer from the front laboratory bench.
17. Wash your comet chip strips by adding enough neutralization buffer to cover and incubate for 5 min at room temperature.
    • Repeat this step a total of 3x.
18. Add the SYBR gold DNA stain to your comet chip strips and carefully move it to the 4 °C cooler.

The teaching faculty will image your comet chip strips after ~16 hr and provide the images to you in the next laboratory section.

Reagents

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