20.109(S14):Data analysis (Day7)
Contents
Introduction
Topic 1: document some of the lecture info on NHEJ key players, leading to
Topic 2: more about C401 inhibitor, and
Topic 3: more about colony-forming assay and staining approach
Topic 4: but the most interesting/fun will be flow analysis: mean vs median choice; breaking down the Day 5 equation a bit more
Oh, and just a word about the MCS/GC/etc. issue for context
Protocols
Part 1: Stain irradiated cell colonies
Option to do it on M3D1 if they want to grow longer for bigger colonies?
All in main lab: rinse w/ 2mL pre-warmed PBS, add 2 mL Coomassie for 1 hr w/shaking, save it afterward, rinse w/PBS again, let dry a little bit, then count colonies right away. (Suggestions for counting approach and how to decide which ones pass the threshold.)
Part 2: Flow cytometry analysis
Overview:
- You will begin by looking at images from the instructor samples to learn how to read the plots and summary statistics.
- Next you will peek at your own images and form preliminary expectations about your data set.
- Finally, you will work in Excel to precisely calculate the NHEJ repair value for each of your three conditions.
Protocol:
- On one of the lab computers, double-click on the FACS server shortcut.
- Alternatively, on your own computer access 18.159.2.11 directly. Ask your instructors for the username and password.
- Go to the April 2014 folder, then to Agi Stachowiak. Copy over both the T/R and W/F image sets to your laptop: the filenames begin "analysis-images" and only the dates differ.
- Copy over just your own day of statistics, unless you really want access to all of the raw data in your back pocket: the .csv filenames begin "analysis-statistics" and only the dates differ.
- The instructor samples are listed in the table below. From this table, and from the T/R and W/F image sets, try to address the questions below.
- Background. The scatter data is used to make gate P3, which should consist primarily of live, single cells. From the cells gated in P3, two sub-gates are made that capture all GFP-positive cells ("Green cells" gate) and all BFP-positive cells ("Blue cells" gate). Both singly and doubly positive cells are included in each gate. It is important to read the "% Parent" statistics: these indicate XFP-positive cells as a percentage of all the cells in P3. The "% Total" statistics include debris, aggregates, and clearly dead cells!
- What percent Green cells are in the mock sample on each day? What about Blue cells?
- What percent of singly-transfected cells express GFP? Do within-day and cross-day replicates agree well or not?
- What percent of singly-transfected cells express BFP? Do within-day and cross-day replicates agree well or not?
- What percent of co-transfected cells express both GFP and BFP? Just GFP? Just BFP? How about that replicate agreement?
- Does ethanol appear to affect scatter profiles? What about GFP, BFP, or co-expression?
- What NHEJ repair value do you calculate for Zac's original BFP plasmid, using the T/R no-ethanol K1 data? Try this calculation by hand. Later, you can include this data as a check on your Excel worksheet. The value you should calculate is X.
First look at instructor mock/single/etc. samples plus own plots.
Then move to stats. Save-as Excel sheet, keep just what they need.
Prepare separate sheet with columns/calculations of interest.
Finally, copy in the data.
Add all B/G ratios and NHEJ ratios group Excel worksheet somehow?
For next time
Methods, as promised.
Reagent list
write something here or not accessible to edit