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

Introduction

Protocols

Part 1: Examine CometChip loading experiments

In a group discussion with the teaching faculty, you will assess the results of the class data from the CometChip loading experiments. The goal here is to determine which cell number and loading time to use when preparing your CometChip 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 CometChips 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 CometChip, you will induce DNA damage 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 ~10 M concentration.
   • You will add 100 μL per well to induce DNA damage.
   • Calculate dilutions that result in doses of 0, 10, 20, 40, 60, and 80 μM.
2. 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.
3. Obtain an aliquot of H₂O₂ and prepare the doses that you calculated in Step #1 using 1x PBS as the diluent in a 12-well reservoir as discussed in prelab.
   • Be sure to keep the H₂O₂ on ice at all times!
4. Using a multi-channel pipet, add the appropriate treatment doses of H₂O₂ to each well of your CometChip.
   • The 0 μM treatment should be added to the wells at the far left and the 80 μM treatment should be added to the wells at the far right of the CometChip.
5. Carefully transport your CometChip to the 4 °C cooler and incubate for exactly 20 min.
6. Retrieve your CometChip from the 4 °C cooler and quickly remove the bottomless 96-well plate while holding the CometChip 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 CometChip using 1x PBS as done during the cell loading procedure.
   • Complete a total of 3 washes.

DNA damage inducer: MMS
Please be very careful when using MMS as it is a mutagen. You should always wear flock-lined 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 ~12 M 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 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.
3. Obtain an aliquot of MMS and prepare the doses that you calculated in Step #1 using 1x PBS as the diluent in a 12-well reservoir as discussed in prelab.
   • Work in the chemical fume hood when preparing MMS solutions!
4. Using a multi-channel pipet, add the appropriate treatment doses of MMS to each well of your CometChip.
   • 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 CometChip.
5. Carefully transport your CometChip to the 37 °C incubator for exactly 30 min.
6. Retrieve your CometChip from the 37 °C incubator and quickly remove the bottomless 96-well plate while holding the CometChip 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 CometChip using 1x PBS as done during the cell loading procedure.
   • Complete a total of 3 washes.

Part 3: Complete CometChip assay

1. Obtain a dish from the front laboratory bench and an aliquot of alkaline lysis solution.
2. Place your CometChip in the dish and add the alkaline lysis solution.
   • Be sure the CometChip is completely submerged and not floating.
3. Move the dish with your CometChip to the 4 °C cooler and incubate for 1 h.
4. Remove your CometChip from the lysis solution and use a kimwipe to dry the gelbond side.
5. Carefully transport your CometChip to the gel electrophoresis station in the 4 °C cold room.
   • The teaching faculty will escort you.
6. Place your CometChip 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 on the tape strips and lightly press down to ensure it is secure.
7. Add enough of the alkaline electrophoresis buffer to the gel electrophoresis box to cover your CometChip.
8. Your CometChip will incubate in the electrophoresis buffer for 40 min to promote unwinding of the DNA.
9. 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.
10. The electrophoresis will continue for 30 min.
11. Carefully remove your CometChip from the electrophoresis box and place it in a dish.
12. Obtain an aliquot of neutralization buffer from the front laboratory bench.
13. Wash your CometChip by adding enough neutralization buffer to cover and incubate for 5 min at room temperature.
    • Repeat this step a total of 3 washes.
14. Add the SYBR gold DNA stain to your CometChip and carefully move it to the 4 °C cooler.

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

Reagents

Navigation links

Next day: Assess DNA repair variability with comet chip