Difference between revisions of "20.109(F17):Evaluate cell loading results (Day3)"

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(Part 3: Prepare CometChips to test biochemical factors)
(Reagents list)
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==Reagents list==
 
==Reagents list==
 +
'''CometChip:'''
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*agar, normal melting point (Invitrogen)
 +
*phosphate buffered saline
 +
*GelBond film (Lonza)
 +
*1 well dish (VWR)
  
 
==Navigation links==
 
==Navigation links==

Revision as of 19:04, 3 September 2017

20.109(F17): Laboratory Fundamentals of Biological Engineering

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Fa17 Schedule        Announcements        Assignments        Homework        Communication
       1. Measuring Genomic Instability        2. Manipulating Metabolism        3. Engineering Biomaterials              

Introduction

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 designing effective figures and captions.

Part 2: Image cell loading experiment

In the previous laboratory session you used the light microscope in the teaching laboratory to image your CometChip and determine the number of wells that were loaded and how many cells were in each of the wells. Today you will use a fluorescent microscope in the Engelward Laboratory to confirm that enough cells were loaded to allow for the visualization of the DNA in the microwells. This will also allow you to practice the image processing steps that you will use to examine your next CometChip experiment.

The teaching faculty will take one team at a time to the microscope in the Engelward Laboratory and demonstrate how the CometChip wells are imaged. While you are waiting for your turn, complete the image processing steps below with the images collected by the teaching faculty and posted to the Discussion tab of the Overview page. Note: due to time constraints the teaching faculty imaged your entire CometChip and those images should be used for this exercise, the images you collect today are for demonstration purposes only.

Complete stacking step and use images to make generalizations concerning DNA signal??

Part 3: Prepare CometChips to test biochemical factors

  1. Obtain a sheet of gelbond film from the laboratory bench at the front of the room. The paper is protecting the hydrophilic side of the gelbond film.
    • Be sure to keep the paper associated with the gelbond film so you know which side is which.
  2. Use the ruler in your team drawer to draw a 5 x 9 cm rectangle on the hydrophobic side of the gelbond film.
    GelBond marking for CometChip
    • Note: you are writing on what will be the bottom of the CometChip and may want to write backwards so the labels are clear when you look at the top of your CometChip.
  3. Prepare 20 mL of 1% normal melting point (NMP) agarose. Be careful as the agarose solution will be very hot!
    • Calculate the amount of NMP agarose powder needed for a 1% w/v solution. Check your math with the teaching faculty before you continue.
    • Obtain a small milk bottle from the front bench.
    • Weigh out the appropriate amount of NMP agarose and add it to the milk bottle.
    • Use a cylinder to measure 20 mL of 1x PBS and add it to the milk bottle with the NMP agarose powder.
    • Swirl to mix.
    • To melt the NMP agarose, microwave the solution for 20 seconds, swirl, then microwave for 3-second intervals until all crystals are in solutions. After each interval, remove the milk bottle and gently swirl while checking for unmelted agarose crystals. It is important that the solution does NOT boil as you will lose water to evaporation and the density of the agarose will be altered. If your solution starts to boil, immediately remove it from the microwave and gently swirl.
    • When no more crystals are visible in the solution take the milk bottle to your bench.
  4. Obtain a small rectangle dish and the CometChip 'stamp' from the front bench.
  5. Add 2.5 mL of the agarose solution to the small dish, then quickly place the gelbond film in the dish with the marked hydrophobic side down.
    • Use a P1000 blue pipet tip to gently tap the gelbond film in order to remove any air bubble as well as ensure that it lays very flat in the dish.
  6. Add 13 mL of the agarose solution on top of the gelbond film.
  7. Slowly place the CometChip stamp on top of the agarose.
    • Lower the bottom left of the stamp first, then slowly allow the stamp to 'roll' into the agarose. Be sure to leave the top right corner of the small dish accessible.
    • Be careful not to introduce bubbles into the agarose and work quickly as the agarose will solidify as it cools.
  8. Allow the agarose to solidy, undisturbed, on your bench for 30 min.
  9. Add ~5 mL of 1x PBS to the small dish that contains your agarose CometChip.
    • Pipet in the 1x PBS using the accessible corner.
  10. Slowly pull from one corner of the stamp to lift it away from your CometChip in the dish.
    • If the CometChip sticks to the stamp, carefully peel it off using tweezers.
    • Discard the PBS in the sink.
  11. Remove excess agarose from the perimeter of your CometChip using a razor blade.
  12. Clean the agarose from the bottom of your CometChip (gelbond side) using a Kimwipe.
  13. Place your CometChip in the small dish containing 1x PBS for storage at 4 °C until next time.
    • Be sure the chip is completely submerged.

Each group needs two chips, time for groups to make two or should teaching faculty make one?

Part 4: Determine cell loading number for subsequent experiments

In a group discussion with the teaching faculty, you will assess the results of the class data from the CometChip loading experiments. The goal here is to determine which cell number and loading time to use when preparing your CometChip for the tests below. Use the data you collected concerning the following: 1. number of wells loaded, 2. number of cells per well, and 3. strength of DNA signal.

Be sure to include notes on the discussion and the values for cell number and loading time that you will use in your notebook!

Reagents list

CometChip:

  • agar, normal melting point (Invitrogen)
  • phosphate buffered saline
  • GelBond film (Lonza)
  • 1 well dish (VWR)

Navigation links

Next day: Test role of biochemical factors in genomic stability

Previous day: Develop experiment to optimize cell loading