Difference between revisions of "20.109(F16):Test comet chip loading variables (Day2)"

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(→‎Part 1: Design experiment to optimize comet chip loading)
(→‎Part 1: Design experiment to optimize comet chip loading)
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You will design two experiments that test variables associated with loading cells into the microwells of your comet chip.  In the first experiment you will consider the role of cell number.  Specifically, how many cells should be added to each well to ensure the majority of the microwells are loaded?  In addition, how many cells should be loaded into each microwell?  The second experiment will interrogate the length of time needed for loading.  In the comet chip that you prepared during the previous laboratory class there is space for you to complete two experiments, each with two conditions.
 
You will design two experiments that test variables associated with loading cells into the microwells of your comet chip.  In the first experiment you will consider the role of cell number.  Specifically, how many cells should be added to each well to ensure the majority of the microwells are loaded?  In addition, how many cells should be loaded into each microwell?  The second experiment will interrogate the length of time needed for loading.  In the comet chip that you prepared during the previous laboratory class there is space for you to complete two experiments, each with two conditions.
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[[File:Fa16 M1D2 comet chip diagram.png|thumb|250px|right|Comet chip schematic for loading variables experiments.]]In the comet chip that you prepared during the previous laboratory class there is space for you to complete two experiments, each with two conditions.
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[[File:Fa16 M1D2 comet chip diagram.png|thumb|250px|right|Comet chip schematic for loading variables experiments.]]In the comet chip that you prepared during the previous laboratory class there is space for you to complete two experiments, each with two conditions.  Your experiments will address the questions above and the conditions will provide data that will, hopefully, answer your research questions.
  
 
'''Experiment 1: Cell number'''<br>
 
'''Experiment 1: Cell number'''<br>
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#How many cells should be added to each well such that the desired number of cells are loaded into the microwells?
 
#How many cells should be added to each well such that the desired number of cells are loaded into the microwells?
 
#*Consider the likelihood that every cell you add to the well will fall into a microwell.  Perhaps calculate the surface area of the bottom of a well and compare this to the size of the cells as you consider this question.
 
#*Consider the likelihood that every cell you add to the well will fall into a microwell.  Perhaps calculate the surface area of the bottom of a well and compare this to the size of the cells as you consider this question.
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#*Now that you have an idea as to the number of cells that are ideal for loading, consider how you will test this in your experiment.   
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#*Now that you have an idea as to the number of cells that are ideal for loading, consider the conditions you will use in your experiment.
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#**If you and your partner are in agreement on how many cells are ideal, you can load the ideal number of cells for Condition A and then either too few or too many for Condition B.  Or you can load slightly more than the ideal number of cells for Condition A and slightly less for Condition B.
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#**If you and your partner are not in agreement, you can each load the number of cells you determine to be ideal as the two Conditions.
  
 
'''Experiment 2:  Loading time'''
 
'''Experiment 2:  Loading time'''

Revision as of 19:18, 30 June 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

experimental design...


Protocols

Part 1: Design experiment to optimize comet chip loading

You will design two experiments that test variables associated with loading cells into the microwells of your comet chip. In the first experiment you will consider the role of cell number. Specifically, how many cells should be added to each well to ensure the majority of the microwells are loaded? In addition, how many cells should be loaded into each microwell? The second experiment will interrogate the length of time needed for loading. In the comet chip that you prepared during the previous laboratory class there is space for you to complete two experiments, each with two conditions.

Comet chip schematic for loading variables experiments.
In the comet chip that you prepared during the previous laboratory class there is space for you to complete two experiments, each with two conditions. Your experiments will address the questions above and the conditions will provide data that will, hopefully, answer your research questions.

Experiment 1: Cell number

Distinction between 'well' and 'microwell' for the comet chip assay.
This first experiment has two questions that should be considered as you discuss the variables you will test with your partner. Before moving on to these questions, it is important to differentiate between the terms 'well' and 'microwell' for your experiments. Bottomless 96-well plates are placed on top of the agarose comet chip to create the wells for the comet chip assay (panel A). This enables researchers to control which cells are exposed to which treatment. The microwells were stamped into the agarose when you made your comet chip (panel B and see M1D1 Part 2, Step #7). Within each well is XX microwells, which are X x X &u;m.
  1. How many cells should be loaded into each microwell?
    • Consider the amount of DNA that is carried by a single mammalian cell and the detection limit provided by the SYBR gold DNA stain that will be used in your experiments. Also, use the data you collected during the Orientation exercise to determine how many cells can fit into a single microwell based on the dimensions provided above.
    • When you know how many cells you want to load into each microwell, move on to the next question.
  1. How many cells should be added to each well such that the desired number of cells are loaded into the microwells?
    • Consider the likelihood that every cell you add to the well will fall into a microwell. Perhaps calculate the surface area of the bottom of a well and compare this to the size of the cells as you consider this question.
    • Now that you have an idea as to the number of cells that are ideal for loading, consider the conditions you will use in your experiment.
      • If you and your partner are in agreement on how many cells are ideal, you can load the ideal number of cells for Condition A and then either too few or too many for Condition B. Or you can load slightly more than the ideal number of cells for Condition A and slightly less for Condition B.
      • If you and your partner are not in agreement, you can each load the number of cells you determine to be ideal as the two Conditions.

Experiment 2: Loading time

Part 2: Load comet chip

Reagents

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