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

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#How many cells should be loaded into each microwell?
 
#How many cells should be loaded into each microwell?
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#*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.
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#*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.
 
#*
 
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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?
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#*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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'''Experiment 2:  Loading time'''
 
'''Experiment 2:  Loading time'''

Revision as of 18:46, 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.

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.
  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.

Experiment 2: Loading time

Part 2: Load comet chip

Reagents

Navigation links

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