20.109(F16):Test comet chip assay variables (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: Design experiment to optimize comet chip assay

In your experiments from the previous laboratory session, you completed tests to determine the best cell number and loading time to use in the comet chip assay. Today you will examine variables that affect the output. Specifically, you will test conditions that affect the robustness and reproducibility of the comet tails. Remember, the comet tail length is used to measure DNA damage in the comet chip assay.

The two variables that you will test are the length of unwinding time and the length of gel electrophoresis time. Unlike your previous experiment, each team will examine only one experiment with two conditions. Read through your notes regarding the role of unwinding time and electrophoresis time to determine which variable you want to examine today.

As you design your experiment, consider the notes below.

Experiment option 1: Unwinding time
To unwind the DNA, your comet chip will incubate in an alkaline electrophoresis buffer. If your unwinding time is too short, the DNA will not be in a relaxed form and remain in the packed chromatin resulting in little to no DNA migration during the electrophoresis step. If your unwinding time is too long, the DNA may become damaged by the NaOH in the buffer and give 'false positive' results.

Experiment option 2: Electrophoresis time
After unwinding, an electric current is applied to separate the damaged DNA and create the comet tail. If your electrophoresis time is too short, the DNA will not migrate far from the microwell and be difficult to measure. If your electrophoresis time is too long, the DNA may migrate into downstream microwells and make the results impossible to interpret.

Record the experimental variable that you will examine and the two conditions you will test in your notebook. When you are ready to proceed, alert the teaching faculty.

Part 3: Complete comet chip assay

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.

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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