20.109(F16):Asses DNA repair variability with comet chip (Day4)

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20.109(F16): Laboratory Fundamentals of Biological Engineering

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Schedule Fall 2016        Announcements        Assignments        Homework        Communication
       1. Measuring Genomic Instability        2. Manipulating Metabolism        3. Engineering Biomaterials              

Introduction

During the previous laboratory section, you used the CometChip assay to measure DNA damage in response to chemical treatments. The output can be described as the amount of DNA damage generated by differing levels of a treatment. The CometChip assay can also be used to measure the repair capacity of a particular cell population. In the experiment you complete today, you will use a set level of treatment, specifically a single concentration of H2O2, then examine repair over a timecourse.

The capacity of a cell line to repair DNA damage raises an interesting question concerning genetic variability – are all cells able to repair base lesions equally? If we add another layer of complexity, the question can be directed toward higher-level organisms – do all humans have the same capacity for repairing base pair lesions? We will address the more complex of the two questions by using the CometChip assay to measure DNA damage repair in three cell lines from the Coriell Institute for Medical Research. The Coriell cells we will use are lymphoblasts collected from healthy individuals.

By examining DNA repair in healthy individuals, researchers can interrogate the normal range of repair capacity and, potentially, identify persons who are at risk for developing cancer. A strategy for identifying individuals at risk would be beneficial in early detection, treatment, and prevention.

Protocols

Part 1: Examine CometChip data for H2O2 induced damage

In a group discussion with the teaching faculty, you will assess the results of the class data from the DNA damage CometChip experiments. The goal here is to determine what concentration of H2O2 to use when preparing your CometChip for the tests below.

Be sure to include notes on the discussion and the H2O2 concentration that you will use in your notebook!

Part 2: Load cells and induce DNA damage

For this experiment everyone will use H2O2 to induce DNA damage in three Coriell cell lines to assess the DNA repair capacity variability in healthy individuals. Use the following plate map to as you complete the below steps:

Plate map for CometChip repair assay. The labels on the right refer to the Coriell cell lines and the labels on the bottom refer to length of time allotted for recovery following exposure to H2O2. Grey wells will be empty for technical reasons.
  1. Load the CometChip prepared by the teaching faculty using the procedure from M1D2; however, please note the procedure for 'trapping' the cells into the microwells will be as follows:
    • Retrieve one tube of molten 1% low melting point (LMP) agarose from the front laboratory bench.
      • You will need to work quickly from this point as the 1% LMP agarose will solidify as it cools.
      • Using the P1000, pipet up 1000 μL.
      • Hold the pipet tip over the top left well of your CometChip and as you expel the agarose move the pipet tip from left to right. Complete this process for every row of your CometChip.
      • Leave your CometChip undisturbed on your bench for 3 min, then move it to the 4 °C cooler for 3 min.
    • For the cell number you will load 25,000 cells per well and for the loading time you will use 30 min.
  2. 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 mM concentration.
    • You will add 100 μL of H2O2 at the concentration determine in Part 1 per well to induce DNA damage.
  3. 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.
  4. Obtain an aliquot of H2O2 and prepare the concentration that you calculated in Step #2 using 1x PBS as the diluent.
    • Be sure to keep the H2O2 on ice at all times!
  5. Add the diluted H2O2 to each well of your CometChip that contains cells, using a reservoir and a multi-channel pipet.
  6. Carefully transport your CometChip to the 4 °C cooler and incubate for exactly 20 min.
  7. Retrieve your CometChip from the 4 °C cooler and quickly remove the bottomless 96-well plate.
  8. Wash your CometChip using 1x PBS as done during the cell loading procedure.
    • Hold your CometChip such that the cells will 'run' over wells that contain a different cell line (i.e. hold the glass plate such that the rows are up and down).
    • Complete a total of 3 washes.

Part 3: Use CometChip to assess repair capacity

  1. Obtain 2 dishes, an aliquot of alkaline lysis buffer, and an aliquot of pre-warmed media from the front laboratory bench.
    • Dispense the lysis buffer and media each into one of the dishes.
  2. Carefully cut your CometChip into strips wherein the 6 wells with the same recovery time remain together.
    • Cut through the center of the empty wells to ensure you do not disrupt the experimental wells.
  3. Place the 'No drug' and '0 min' strips directly into the alkaline lysis solution at 4 °C.
  4. Place the remaining strips into the media and carefully move the dish to the 37 °C incubator.
    • Be sure to set a timer!
  5. After 20 min, retrieve your dish from the 37 °C incubator and remove the '20 min' strip.
    • Add the '20 min' strip to the lysis solution.
  6. Complete Step #5 after 40 min and 60 min, transferring the appropriate strip to the lysis solution.
  7. Move the dish with your CometChip strips to the 4 °C cooler and incubate for 1 h.
  8. Remove your CometChip strips from the lysis solution and use a kimwipe to dry the gelbond side.
  9. Carefully transport your CometChip strips to the gel electrophoresis station in the 4 °C cold room.
    • The teaching faculty will escort you.
  10. Place your CometChip strips 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 strips on the tape strips and lightly press down to ensure it is secure.
  11. Add enough of the alkaline electrophoresis buffer to the gel electrophoresis box to cover your CometChip strips.
  12. Your CometChip strips will incubate in the electrophoresis buffer for 40 min to promote unwinding of the DNA.
  13. 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.
  14. The electrophoresis will continue for 30 min.
  15. Carefully remove your CometChip strips from the electrophoresis box and place it in a dish.
  16. Obtain an aliquot of neutralization buffer from the front laboratory bench.
  17. Wash your CometChip strips by adding enough neutralization buffer to cover and incubate for 5 min at room temperature.
    • Repeat this step a total of 3x.
  18. Add the SYBR gold DNA stain to your CometChip strips and carefully move it to the 4 °C cooler.

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

Reagents list

CometChip:

  • agar, normal melting point (Invitrogen)
  • phosphate buffered saline
  • GelBond film (Lonza)
  • 1 well dish (VWR)
  • agar, low melting point (Invitrogen)
  • bottomless 96-well plates (VWR)

Cell culture:

  • RPMI 1640 (Invitrogen)
    • with 10% fetal bovine serum
    • and 1% penicillin/streptomycin
  • Trypan Blue
  • H2O2: hydrogen peroxide (Sigma Aldrich)

Alkaline lysis solution: (Sigma Aldrich)

  • 2.5 M NaCl
  • 100 mM Na2EDTA
  • 10 mM Tris
  • pH 10

Alkaline electrophoresis solution: (Sigma Aldrich)

  • 0.3 MNaOH
  • 1 mM Na2EDTA
  • pH 13.5

Neutralization buffer: (Sigma Aldrich)

  • 0.4 M Tris
  • pH 7.5

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