20.109(F17):Complete biochemical experiment (Day5)

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20.109(F17): Laboratory Fundamentals of Biological Engineering

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       1. Measuring Genomic Instability        2. Manipulating Metabolism        3. Engineering Biomaterials              


Introduction

To this point in the module we have been using the CometChip assay to study DNA damage in the context of the BER pathway. Specifically, we considered the role of oxidative stress in generating base lesions. It is important to remember that the data collected using the CometChip assay is not specific to base lesions and that the DNA damage observed may be the result of various types of DNA lesions, including base excisions, abasic sites, strand breaks, and crosslinks. The experimental work you complete in the next three laboratory classes will focus on a technique used to measure DNA double-strand breaks.

H2AX is phosphorylated in response to DNA double-strand breaks.
In eukaryotes, including humans, DNA is tightly wound around histone groups. H2AX is a member of the core group of histones that contributes to nucleosome formation and DNA structure. When a DNA double-strand break is introduced into the genome, the H2AX histones near the break are phosphorylated by the ATM kinase at residue Ser-139. Upon phosphorylation H2AX is referred to as gamma-H2AX. Given that only H2AX histones near the site of DNA damage are phosphorylated, γH2AX is a useful target when determining the abundance and location of double-strand breaks.

In your γH2AX assay experiment, you will assess the effects of H2O2 and MMS on double-strand break abundance in the cell lines used for the CometChip assay. In your analysis you will interrogate the consistency of the results from these two approaches. Furthermore, you will craft a hypothesis concerning the repair efficiency of the cell lines and use the γH2AX assay to test your research question.

Protocols

Part 1: BE Communication Lab workshop

Our communication instructors, Dr. Sean Clarke and Dr. Prerna Bhargava, will join us today for a workshop on writing impactful abstracts and titles.

Part 2: Complete enzyme treatments for biochemical test experiments

Each team will use the enzyme that corresponds to the chemical treatment selected during the previous laboratory session.

Follow the appropriate protocol for damage induction based on the chemical you selected for your experiment.

DNA damaging agent: H2O2

One CometChip will be a 'no enzyme treatment' control and the other will be the 'treatment' experimental with enzyme. Be sure to label and keep track of which chip is which.

  1. While you were away, the lysis buffer was removed and the CometChips were submerged in PBS. The CometChips were then equalized in 1x complete Fpg reaction buffer.
  2. Place the CometChips at an angle on paper towels (by leaning one side of the chip against the edge of the dish and the other side against the paper towel) and allow to dry for 3 min.
  3. Obtain a glass plate, bottomless 96-well plate, and four binder clips for each CometChip and assemble a 'sandwich' as done previously.
    • Ensure that the 96 well plate is oriented properly
  4. Obtain one aliquot of 1x complete Fpg reaction buffer from front bench.
  5. Obtain one aliquot of Fpg enzyme solution (already diluted 1:10,000 in 2.5 mL of 1X complete Fpg reaction buffer) from front bench.
  6. In the 'no enzyme treatment' control CometChip, add 50 μL of 1X complete Fpg reaction buffer to each well that was loaded and treated during the previous laboratory session. Use your group's P200 to aliquot this buffer in the macrowells.
  7. In the 'treatment' experimental CometChip, add 50 μL of Fpg enzyme solution to each well that was loaded and treated during the previous laboratory session. Use a single well dish and multiwell pipette to aliquot the buffer and enzyme solution in the macrowells.
  8. Incubate both CometChips at 37 °C for 20 min.
    • Meanwhile, rinse out the dishes with deionized water in the sink so you can use them for the next step.
  9. To stop the reaction, remove the bottomless 96-well plate and submerge the CometChips in 40 mL of cold 1X PBS.
    • Place chip into dish and bring dish to to cold room (ask instructor).
    • Obtain 40 mL aliquot of PBS in cold room and pour into the corner of the dish.
  10. Immediately transfer the CometChips to 30 mL of cold alkaline unwinding buffer.
    • Carefully pour the PBS in the cold room sink.
    • Carefully pour one aliquot (30 mL) of alkaline unwinding buffer into the dish and ensure the chip is submerged.
  11. Incubate for 1 h in the cold room.

DNA damaging agent: MMS

One CometChip will be a 'no enzyme treatment' control and the other will be the 'treatment' experimental with enzyme. Be sure to label and keep track of which chip is which.

  1. While you were away, the lysis buffer was removed and the CometChips were submerged in PBS. The CometChips were then equalized in 1x complete hAAG reaction buffer.
  2. Place the CometChips at an angle on paper towels (by leaning one side of the chip against the edge of the dish and the other side against the paper towel) and allow to dry for 3 min.
  3. Obtain a glass plate, bottomless 96-well plate, and four binder clips for each CometChip and assemble a 'sandwich' as done previously.
    • Ensure that the 96 well plate is oriented properly
  4. Obtain one aliquot of 1x complete hAAG reaction buffer from front bench.
  5. Obtain one aliquot of hAAG enzyme solution (already diluted 1:10,000 in 2.5 mL of 1X complete hAAG reaction buffer) from front bench.
  6. In the 'no enzyme treatment' control CometChip, add 50 μL of 1X complete hAAG reaction buffer to each well that was loaded and treated during the previous laboratory session. Use your group's P200 to aliquot this buffer in the macrowells.
  7. In the 'treatment' experimental CometChip, add 50 μL of hAAG enzyme solution to each well that was loaded and treated during the previous laboratory session. Use a single well dish and multiwell pipette to aliquot the buffer and enzyme solution in the macrowells.
  8. Incubate both CometChips at 37 °C for 15 min.
    • Meanwhile, rinse out the dishes with deionized water in the sink so you can use them for the next step.
  9. To stop the reaction, remove the bottomless 96-well plate and submerge the CometChips in 40 mL of cold 1X PBS.
    • Place chip into dish and bring dish to to cold room (ask instructor).
    • Obtain 40 mL aliquot of PBS in cold room and pour into the corner of the dish.
  10. Immediately transfer the CometChips to 30 mL of cold alkaline unwinding buffer.
    • Carefully pour the PBS in the cold room sink.
    • Carefully pour one aliquot (30 mL) of alkaline unwinding buffer into the dish and ensure the chip is submerged.
  11. Incubate for 1 h in the cold room.

Part 3: Separate CometChip 'tails' using gel electrophoresis

  1. Remove your CometChip from the alkaline unwinding buffer and use a kimwipe to dry the GelBond side.
  2. Carefully move your CometChip to the gel electrophoresis station in the 4 °C cold room.
  3. 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 with a pipet tip to ensure it is secure.
  4. Add enough of the alkaline electrophoresis buffer to the gel electrophoresis box to cover your CometChip.
  5. 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.
  6. The electrophoresis will continue for 30 min (at 16 V, or 1 V/cm).
  7. Carefully remove your CometChip from the electrophoresis box and place it in a dish.
  8. Obtain an aliquot of neutralization buffer from the front laboratory bench.
  9. Wash your CometChip by adding enough neutralization buffer to cover (~10-12mL) and incubate for 5 min at room temperature.
    • Repeat this step a total of 3 washes.
  10. 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 and provide the images to you in the next laboratory section.

Part 4: Treat cells for sub-nuclear foci assay

For timing reasons, the teaching faculty seeded 12-well plates with wild-type and mutant cells that you will treat with the same chemical you used for the biochemical testing experiment. Each group will work with two plates: one will be used to assess the level of damage following treatment with the DNA damaging agent and the second will be used to assess repair following treatment.

Before you enter the tissue culture room, sign up for the treatment concentrations and recovery time that you will use in your experiment and confirm your calculations with the teaching faculty. Each team will test two concentrations (in addition to completing 'no treatment' control samples).

  1. Choose which concentrations of DNA damaging agent you will test.
    • For H2O2, select from 5, 10, 15, 20, and 25 μM.
    • For MMS, select from 0.1, 0.2, 0.5, 6, 8 mM.
  2. Calculate your dilutions such that you have 4 mL of pure DMEM media (FBS- and antibiotic-free) containing DNA damaging agent for each concentration.
    • For H2O2, the stock concentration is 10 M. The teaching faculty prepared a 100 μM solution that will be used to prepare your treatment concentrations.
    • For MMS, the stock concentration is 12 M. The teaching faculty prepared a 12 mM solution that will be used to prepare your treatment concentrations.
  3. Alert the teaching faculty when you are ready to enter the tissue culture room.
  4. Upon entering the tissue culture room, ready your tissue culture hood with the supplies you will need to complete the experimental procedures.
  5. Prepare your DNA damaging agents according to the calculations you completed in Step #2.
  6. Retrieve the two 12-well plates that were seeded for you by the teaching faculty from the 37 °C incubator.
  7. Add 450 μL of either DMEM or DMEM + damaging agent to the appropriate wells according to the plate map below.
    Plate map for γH2AX assay
  8. Incubate at 37 °C for 30 min.
  9. Retrieve your 12-well plates and collect the media in the correct waste stream using a P1000 pipet.
  10. Immediately add 500 μL of 4% paraformaldehyde to one of your 12-well plates to fix the cells.
  11. Incubate at room temperature for 10 min.
  12. During this incubation, add 500 μL of DMEM (containing FBS and antiobiotics) to the remaining 12-well plate and move it to the 37 ° incubator for recovery.
  13. Collect the 4% paraformaldehyde in the correct waste stream using a P1000 pipet.
  14. Wash with 500 μL of 1X PBS.
    • Add 1X PBS then remove using a P1000 pipet, and collect it in the correct waste stream.
    • Complete a total of 2 times.

Reagents list

H2AX assay

  • DMEM (Gibco)
    • with 10% fetal bovine serum (Atlanta Biologicals)
    • 100X antibiotic solution (Gibco)
      • 10,000 U/mL Penicillin
      • 10,000 U/mL Streptomycin
  • MMS: methyl methanesulfonate (Sigma Aldrich)
  • H2O2: hydrogen peroxide (Sigma Aldrich)
  • paraformaldehyde 4% (VWR)

Biochemical testing

  • Fpg enzyme (New England Biolabs)
  • hAAG enzyme (New England Biolabs)
  • 1x complete Fpg reaction buffer (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA)
  • 1x complete hAAG reaction buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100)
  • Alkaline unwinding buffer (a.k.a. Alkaline electrophoresis buffer, 0.3M NaOH, Na2EDTA)

Navigation links

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