Difference between revisions of "20.109(F16):Asses DNA repair variability with comet chip (Day4)"

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(→‎Part 1: Load cells and induce DNA damage)
(→‎Part 1: Load cells and induce DNA damage)
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[[Image:Fa16 M2D4 repair assay plate map.png|thumb|center|500 px|'''Plate map for comet chip 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 the H<sub>2</sub>O<sub>2</sub>.  Grey wells will be empty for technical reasons.]]
 
[[Image:Fa16 M2D4 repair assay plate map.png|thumb|center|500 px|'''Plate map for comet chip 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 the H<sub>2</sub>O<sub>2</sub>.  Grey wells will be empty for technical reasons.]]
  
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You will load comet chips prepared by the teaching faculty using the procedure from [http://engineerbiology.org/wiki/20.109%28F16%29:Test_comet_chip_loading_variables_%28Day2%29#Part_2:_Load_comet_chip M1D2]; however, please note the following differences:
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#You will load comet chips prepared by the teaching faculty using the procedure from [http://engineerbiology.org/wiki/20.109%28F16%29:Test_comet_chip_loading_variables_%28Day2%29#Part_2:_Load_comet_chip M1D2]; however, please note the following differences:
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*You will not complete the lysis incubation immediately following the cell loading, rather this will be done after the DNA damage induction step.
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#*You will not complete the lysis incubation immediately following the cell loading, rather this will be done after the DNA damage induction step.
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*For the cell number you will use 500 K/mL and for the loading time you will use 30 min.
+
#*For the cell number you will use 500 K/mL and for the loading time you will use 30 min.
 +
#While the LMP agar is solidifying at 4 &deg;C, calculate the dilutions of H<sub>2</sub>O<sub>2</sub> that you will use for your experiment.
 +
#*The H<sub>2</sub>O<sub>2</sub> stock solution is at a ~10 M concentration.
 +
#*You will add 100 &mu;L of 50 &mu;M per well to induce DNA damage.
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#Retrieve your comet chip from the 4 &deg;C and carefully replace the bottomless 96-well plate to recreate the wells that you used for cell loading.
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#*Secure the bottomless 96-well plate with binder clips as before.
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#Obtain an aliquot of H<sub>2</sub>O<sub>2</sub> and prepare the concentration that you calculated in Step #2 using 1x PBS as the diluent in a divided [cant remember name of dish] as discussed in prelab.
 +
#*Be sure to keep the H<sub>2</sub>O<sub>2</sub> on ice at all times!
 +
#Add the diluted H<sub>2</sub>O<sub>2</sub> to each well of your comet chip that contains cells.
 +
#Carefully transport your comet chip to the 4 &deg;C cooler and incubate for '''exactly''' 20 min.
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#Retrieve your comet chip from the 4 &deg;C cooler and quickly remove the bottomless 96-well plate.
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#Rinse your comet chip using 1x PBS as done during the cell loading procedure.
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#*Complete a total of 3 washes.
  
 
===Part 2: Use comet chip to assess repair capacity===
 
===Part 2: Use comet chip to assess repair capacity===

Revision as of 17:35, 25 August 2016

20.109(F16): Laboratory Fundamentals of Biological Engineering

Engelward PNAS 2006.png

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

Introduction

Protocols

Part 1: 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 comet chip 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 the H2O2. Grey wells will be empty for technical reasons.
  1. 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 after the DNA damage induction step.
    • For the cell number you will use 500 K/mL 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 M concentration.
    • You will add 100 μL of 50 μM per well to induce DNA damage.
  3. Retrieve your comet chip 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 in a divided [cant remember name of dish] as discussed in prelab.
    • Be sure to keep the H2O2 on ice at all times!
  5. Add the diluted H2O2 to each well of your comet chip that contains cells.
  6. Carefully transport your comet chip to the 4 °C cooler and incubate for exactly 20 min.
  7. Retrieve your comet chip from the 4 °C cooler and quickly remove the bottomless 96-well plate.
  8. Rinse your comet chip using 1x PBS as done during the cell loading procedure.
    • Complete a total of 3 washes.

Part 2: Use comet chip to assess repair capacity

Reagents

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