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 during Step #2 below.
• 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

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.

The following steps will be completed in the main laboratory.

1. Damage cells...
2. Lyse cells...
3. Cut comet chip into segments...
4. Obtain a plastic dish and an aliquot of the alkaline electrophoresis buffer from the front laboratory bench.
5. 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.
6. Remove the comet chip segments from the buffer and use a kimwipe to dry the gelbond side.
7. Carefully transport your comet chip segments to the gel electrophoresis station.
8. 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.
9. Add the alkaline electrophoresis buffer from your plastic dish to the gel electrophoresis box.
10. Carefully carry the gel electrophoresis box to the 4 °C cold room.
    • The teaching faculty will escort you.
11. 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.
12. 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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