Experiment 3: Growth and Cell Division of Wild Type (NC4A2) and Myosin II null mutant (HK321) cells. 

You will set up cultures in Petri dishes & shaking suspension for both NC4A2 and HK321 and compare their growth rates. The ‘wildtype’ axenic NC4A2 strain contains the intact Myosin II heavy chain gene. However, the myosin II gene is ‘knocked out’ in HK321 cells thus translation is aborted and the protein is not expressed. The outcome of this experiment will provide an answer to the question: Is Myosin II required for growth and cell division?

 

A. Setting up Growth Expt for NC4A2 and HK321 null cells.

THURSDAY

1. You will be given HK321 and NC4A2 cells in Petri dishes.  Don’t forget to replace the HL-5 in all of your stock plates after using them for this experiment.

2. Set up NC4A2 and HK321 cell cultures in Petri dishes and flasks at about 5x104 cells/ml. 

3.Titer the cells roughly twice a day over the next 5-7 days.

NOTE: When you are taking samples from the flasks to titer, take the flasks from the shaker, take the sample and add it to the hemocytometer, and put the flask back on the shaker.  Then count the sample.  You do not want to flasks sitting for a long time on the benchtop as the cells will settle to the bottom and become oxygen starved.

For your Lab Report : This is your first formal Lab report!

1.Follow the format with a title, objective, summary of methods, results and discussion. The growth profiles of wildtype and mutant and PI staining results are both incorporated in one lab report.

2.For the results section, make a graph of the growth profile of NC4A2 and HK321 in plates and shaking suspension (linear scaling in both X and Y axes. Also, generate semilog plots for each condition and calculate the generation times for each.

Its up to you how you present your results as long as these data are in your results. These graphs should also be posted in your websites along with your generation times and conclusions.

Lab report and website data uploads for this experiment are due 1 week after you finish collecting data.    

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