20.109(F16):Test comet chip assay variables (Day3)
Contents
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 effect the output. Specifically, you will test conditions that effect 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 variable. 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 you 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
Reagents
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Previous day: Test comet chip loading variables