20.109(S15):Data analysis (Day7)

Introduction

Protocols

Part 1: Visit by Leslie to discuss the Discussion section

We will start the day with a visit by our writing instructor, Leslie, who will talk about the structure of a Discussion section, the only part of a formal research paper you have yet to write.

Part 2: Statistics practice

You may find averages, standard deviations, and t-tests useful when you report on class results.

You can practice the steps below using the male and female heights that we collect during pre-lab lecture.

1. Begin by downloading the following Excel file as a framework to carry out the basic statistical manipulations we discussed in pre-lab lecture. The file is modified from one originally written by Professor Bevin Engelward.
2. Find and plot 95% confidence intervals for the male and female heights.
3. Compare the means of these two populations. At what confidence level (if any) are they different?
   • Would a one-tailed or two-tailed test be more appropriate for this comparison?

Part 3: Stain irradiated cell colonies

Typical growth times for clonogenic assays are at least one week, and if you stain next time your counting task may be made easier by having bigger colonies. On the other hand, you have built-in class time today for analysis, and may want to work on your dose response curve after you complete the FC analysis. The decision is totally up to you and your partner!

1. Briefly observe your irradiated cells on the TC microscopes. Are you able to find some colonies? About how many cells are in some of these small dispersed clusters?
2. Take your plate to the main lab for the remaining steps. First, aspirate the media. You don't need to change the yellow tip between samples if you move from highest [C401] dose to lowest, as that should be the order of least to most colonies.
3. Rinse each well with about 2 mL of pre-warmed PBS.
4. After removing the PBS, use a serological pipet to consistently add 2 mL of Coomassie to each well.
5. Place your plate on the fume hood shaker at 80 rpm for about 1 hour.
6. Repeat the PBS rinse, this time with room temperature buffer.
7. Let the well plate dry for a short time after aspirating the PBS.
   • If you wait a very long time the stain will begin to fade.
8. Finally, count the colonies in each well and document these on today's Talk page.
   • Do your best to apply a consistent standard for threshold colony size and threshold staining intensity. As long as you are consistent, there is not one right answer as to what constitutes a colony.
   • One of your instructors finds it easiest to count colonies by making a dot with a lab marker as she counts each colony, right on the underside of the 6-well plate at the colony location, and then writing down each decade (10, 20, etc.) on the plate as she reaches that number so she doesn't lose her place.
9. Whether today or on your own, you should plot your data by surviving fraction (as described in the Nature Protocols paper linked above) for the Module 2 report. However, you don’t need to perform a curve fit as we are essentially looking for a yes/no answer to the question of whether the inhibitor worked.

Reagent list

• PBS
• Bio-Safe Coomassie Stain (Bio-Rad)
• Mostly your brains!

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