Difference between revisions of "20.109(F16):Measure DNA damage with comet chip (Day3)"

Introduction

Protocols

Part 1: Examine comet chip loading experiments

In a group discussion with the teaching faculty, you will assess the results of the class data from the comet chip loading experiments. The goal here is to determine which cell number and loading time to use when preparing your comet chip for the tests below.

Be sure to include notes on the discussion and the values for cell number and loading time that you will use in your notebook!

Part 2: Induce DNA damage

You will load comet chips 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 and loading time, you will use the values decided on during the group discussion in Part 1.

After you load the cells into the comet chip, you will induce DNA using either H₂O₂ or MMS as assigned by your teaching faculty. Follow the appropriate protocol for damage induction based on the chemical you were assigned to test.

DNA damage inducer: H₂O₂

1. 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 ~10M concentration.
   • You will add 100 μL per well to induce DNA damage.
   • Calculate dilutions that result in doses of 0, 5, 10, 20, 40, 60, and 80 μM.
2. 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.
3. Obtain an aliquot of H₂O₂ and prepare the doses that you calculated in Step #1 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!
4. Add the appropriate treatment doses of H₂O₂ to each well of your comet chip.
   • The 0 μL treatment should be added to the wells at the far left and the 80 μL treatment should be added to the wells at the far right of the comet chip.
5. Carefully transport your comet chip to the 4 °C cooler and incubate for exactly 20 min.
6. Retrieve your comet chip from the 4 °C cooler and quickly remove the bottomless 96-well plate while holding the comet chip at an angle such that the low doses of H₂O₂ 'run' over the wells with high doses.
   • If the high doses of H₂O₂ run over the low dose wells, additional damage may be induced and confound the results.
7. Rinse your comet chip using 1x PBS as done during the cell loading procedure.

DNA damage inducer: MMS
Please be very careful when using MMS as it is a mutagen. You should always wear XX gloves, in addition to the standard laboratory gloves, when using MMS.

1. While the LMP agar is solidifying at 4 °C, calculate the dilutions of MMS that you will use for your experiment.
   • The MMS stock solution is at a ~12M concentration.
   • You will add 100 μL per well to induce DNA damage.
   • Calculate dilutions that result in doses of 0, 0.25, 0.5, 1, 2, 4, and 8 mM.
2. 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.
3. Obtain an aliquot of MMS and prepare the doses that you calculated in Step #1 using 1x PBS as the diluent in a divided [cant remember name of dish] as discussed in prelab.
   • Work in the chemical fume hood when preparing MMS solutions!
4. Add the appropriate treatment doses of MMS to each well of your comet chip.
   • The 0 mM treatment should be added to the wells at the far left and the 8 mM treatment should be added to the wells at the far right of the comet chip.
5. Carefully transport your comet chip to the 37 °C incubator for exactly 30 min.
6. Retrieve your comet chip from the 37 °C incubator and quickly remove the bottomless 96-well plate while holding the comet chip at an angle such that the low doses of MMS 'run' over the wells with high doses.
   • If the high doses of MMS run over the low dose wells, additional damage may be induced and confound the results.
7. Rinse your comet chip using 1x PBS as done during the cell loading procedure.

Part 3: Complete comet chip assay

1. Lyse cells...
2. Obtain a plastic dish and an aliquot of the alkaline electrophoresis buffer from the front laboratory bench.
3. Carefully transfer your comet chip segments to the plastic dish and add the electrophoresis buffer.
   • If your experiment is designed to test unwinding time, include the segment of your comet chip that requires the longer incubation time. Then include the segment of your comet chip that requires the shorter incubation time to the dish when appropriate.
   • If your experiment is designed to test electrophoresis time, incubate both segments of your comet chip for 30 min.
4. Remove the comet chip segments from the buffer and use a kimwipe to dry the gelbond side.
5. Carefully transport your comet chip segments to the gel electrophoresis station.
6. Place each segment 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 on the tape strips and lightly press down to ensure the segments are secure.
7. Add the alkaline electrophoresis buffer from your plastic dish to the gel electrophoresis box.
8. Carefully carry the gel electrophoresis box to the 4 °C cold room.
   • The teaching faculty will escort you.
9. Add enough alkaline electrophoresis buffer to cover the comet chip segments.
   • 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.
10. Separate the damaged DNA with gel electrophoresis.
    • If your experiment is designed to test unwinding time, electrophorese both segments of your comet chip for 30 min.
    • If your experiment is designed to test electrophoresis time, remove the segment of your comet chip that required the shorter separation time when appropriate, using tweezers. Then allow the segment of your comet chip that requires the longer separation time to remain in the electrophoresis box until complete.

When your comet chip assay is complete, move the segments into well-labeled dishes and add enough 1x PBS to cover. Leave the dishes on the front laboratory bench and the teaching faculty will image your comet chip prior to the next laboratory session.

Reagents

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