Difference between revisions of "20.109(F16):Asses DNA repair variability with comet chip (Day4)"

Introduction

Protocols

Part 1: Examine CometChip data for H₂O₂ induced damage

In a group discussion with the teaching faculty, you will assess the results of the class data from the DNA damage CometChip experiments. The goal here is to determine what concentration of H₂O₂ to use when preparing your CometChip for the tests below.

Be sure to include notes on the discussion and the H₂O₂ concentration that you will use in your notebook!

Part 2: 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 CometChip 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 H₂O₂. Grey wells will be empty for technical reasons.

1. Load the CometChip 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 500,000/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 H₂O₂ at the concentration determine in Part 1 per well to induce DNA damage.
3. Retrieve your CometChip 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.
   • Be sure to keep the H₂O₂ on ice at all times!
5. Add the diluted H₂O₂ to each well of your CometChip that contains cells, using a reservoir and a multi-channel pipet.
6. Carefully transport your CometChip to the 4 °C cooler and incubate for exactly 20 min.
7. Retrieve your CometChip from the 4 °C cooler and quickly remove the bottomless 96-well plate.
8. Rinse your CometChip using 1x PBS as done during the cell loading procedure.
   • Complete a total of 3 washes.

Part 3: Use CometChip 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 CometChip 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 '0 min' strips directly into the alkaline lysis solution at 4 °C.
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 CometChip strips to the 4 °C cooler and incubate for 1 h.
8. Remove your CometChip strips from the lysis solution and use a kimwipe to dry the gelbond side.
9. Carefully transport your CometChip strips to the gel electrophoresis station in the 4 °C cold room.
   • The teaching faculty will escort you.
10. Place your CometChip 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 CometChip 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 CometChip strips.
12. Your CometChip 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 CometChip 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 CometChip 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 CometChip strips and carefully move it to the 4 °C cooler.

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

Reagents

Navigation links

Next day: Seed cells for immuno-fluorescence assay