User:Kalkao/notebook

This is the start of my online lab notebook.

Protocols

Making T-broth Agar Plates

Streaking Plates

Making Overnight Suspensions

Freezing Away a Cell Stock

Making Plaques

Lab Notebook

June 14, 2006

• Followed the Making T-broth Agar Plates protocol to make 40 T-broth agar plates. Sri taught me this method.

June 19, 2006

• I used the Streaking Plates protocol to streak one plate with BL21 cells and one plate with IJ1126 cells. Sri taught me this method.

June 20, 2006

• I used the Making Overnight Suspensions protocol to make two overnight suspensions of BL21 cells. Sri taught me this method.

June 21, 2006

• I used the Freezing Away a Cell Stock protocol to make 2 stocks of BL21 cells. These BL21 stocks are located in the bottom shelf of the -80 storage, 2nd row from the top, 2nd column from the left, and are labeled with blue stickers.
• I used the Making Plaques protocol to make 8 plates, which I labeled "Blank A," "Blank B," "10uL A," "10uL B," "100uL A," "100uL B," "200uL A," and "200uL B." I obtained my phage stock (10mL) from Sri's T7+ stock #1. His stock was created 9/28/2006 at 2.4e11 PFU/mL. I labeled this stock T7+ #1. I made 4 approximately 1:100 dilutions of my stock for use in creating plaques. The dilutions are as follows:
  • Dilution 1: 1:100 50uL stock in 5mL T-broth
    • (50uL)(1mL/1000uL)(2.4e11) = (5mL)(Concentration 1)
    • Concentration 1 = 2.4e9 PFU/mL
  • Dilution 2: 1:100 50uL dilution 1 in 5mL T-broth
    • (50uL)(1mL/1000uL)(2.4e9) = (5mL)(Concentration 2)
    • Concentration 2 = 2.4e7 PFU/mL
  • Dilution 3: 1:100 50uL dilution 2 in 5mL T-broth
    • (50uL)(1mL/1000uL)(2.4e7) = (5mL)(Concentration 3)
    • Concentration 3 = 2.4e5 PFU/mL
  • Dilution 4: 1:100 50uL dilution 3 in 5mL T-broth
    • (50uL)(1mL/1000uL)(2.4e5) = (5mL)(Concentration 4)
    • Final Concentration = 2.4e3 PFU/mL
• After incubating the eight plates for 4 hours, Sri and I spotted no plaques. About one and a half hours later, we found a plaque on the "100uL B" plate and the "200uL B" plate. This suggests that we diluted the phage stock too much. The phage stock may have diluted over time, skewing our value for the stock concentration. Also, we noticed that the agar on the plates was not very smooth. This may be due to the soft agar not being warm enough before being poured. We will try keeping the soft agar at a higher temperature before we pour it, to allow it more time to spread out over the plate before it solidifies. Sri walked me through the methods today.

June 22, 2006

• Sri showed me how to make a new T7 stock using the Studier Lysate Prep protocol.
• I used the Making Plaques protocol to make 8 new plates: Blank A, Blank B, 10uL A, 10uL B, 100uL A, 100uL B, 200uL A, and 200uL B. I obtained phage from my T7+ #1 stock. Because Sri and I found very few plaques after 4 1:100 dilutions of my stock yesterday, we decided today to make only 3 1:100 dilutions of my stock and use the final dilution to create plaques. The dilutions are as follows:
  • Dilution 1: 1:100 50uL stock in 5mL T-broth
    • (50uL)(1mL/1000uL)(2.4e11) = (5mL)(Concentration 1)
    • Concentration 1 = 2.4e9 PFU/mL
  • Dilution 2: 1:100 50uL dilution 1 in 5mL T-broth
    • (50uL)(1mL/1000uL)(2.4e9) = (5mL)(Concentration 2)
    • Concentration 2 = 2.4e7 PFU/mL
  • Dilution 3: 1:100 50uL dilution 2 in 5mL T-broth
    • (50uL)(1mL/1000uL)(2.4e7) = (5mL)(Concentration 3)
    • Final Concentration = 2.4e5 PFU/mL
• For the 200uL B plate, after adding the final phage dilution to one of the centrifuge tubes with culture, I forgot to vortex the mixture before adding it to the 3mL of soft agar. I also spilled a bit of the phage/BL21/soft agar mixture while pouring it onto its plate. After pouring the 8 plates, I noticed a black spec in the soft agar of the 10uL B plate.
• These are the plaque counts after 4 hours of incubation:
  • Blank A: 0
  • Blank B: 0
  • 10uL A: 0
  • 10uL B: 2
  • 100uL A: 8
  • 100uL B: 6
  • 200uL A: 21
  • 200uL B: 44
• These numbers seem somewhat inconsistent, especially between the two 200uL plates. Also, there is not enough data to confidently determine the original phage stock concentration. However, if I were to use this data, I would pick the plaque counts from the 100uL plates to calculate an original phage concentration of my stock. From these plaque counts, I arrived at a calculated stock concentration of 7e6 PFU/mL.
• I made 1L of T-broth agar using the Making T-broth Agar Plates protocol.
  • The weight plates had some plastic debris around the sides
  • I placed the T-broth agar in the autoclave at 5:30PM.
• While the T-broth agar was in the autoclave, I made two overnight cultures of BL21 cells, using the Making Overnight Suspensions protocol.
• I removed the T-broth agar at 9:00PM and poured 40 plates. I noticed black specs from the agar in 2 plates and I got one of the plate covers dirty. The black specs may be from the agar, as some of the T-broth agar on the side of the flask was burnt during autoclaving. I also noticed white, stringy debris in many of the plates. It looked like the same plastic debris from the weigh plates.

June 23, 2006

• I checked last night's t-broth agar plates and found no contamination
• I used last night's two overnight cultures find the titer again for the original T7+ #1 stock and also for the new T7+ stock that I created yesterday (I labled this stock T7+ #2). I used the Making Plaques protocol for these titers:
  • To titer the T7+ #1 stock, I decided on making a serial dilution of one 1:100 and three 1:10:
    • Dilution 1: 1:100 50uL stock in 5mL T-broth
      • (50uL)(1mL/1000uL)(7e6) = (5mL)(Concentration 1)
      • Concentration 1 = 7e4 PFU/mL
    • Dilution 2: 1:10 500uL dilution 1 in 4.5mL T-broth
      • (500uL)(1mL/1000uL)(7e4) = (4.5mL)(Concentration 2)
      • Concentration 2 = 7.78e3 PFU/mL
    • Dilution 3: 1:10 500uL dilution 2 in 4.5mL T-broth
      • (500uL)(1mL/1000uL)(7.78e3) = (4.5mL)(Concentration 3)
      • Concentration 3 = 8.64e2 PFU/mL
    • Dilution 4: 1:10 500uL dilution 3 in 4.5mL T-broth
      • (500uL)(1mL/1000uL)(8.64e2) = (4.5mL)(Concentration 4)
      • Final Concentration = 9.6e1 PFU/mL
    • The resulting final dilution should be 10 times more concentrated than last night's final dilution of the T7+ #1 stock. This would allow for more plaques to form on the plates, allowing us to more precisely measure the plaque forming units of the stock. I decided to create plaques using 50uL, 100uL, and 150uL of the final dilution, since I expected the best data to surround these amounts of phage dilution. The plates were labeled Blank A, Blank B, Blank C, 50uL A, 50uL B, 50uL C, 100uL A, 100uL B, 100uL C, 150uL A, 150uL B, and 150uL C.
  • To titer the T7+ #2 stock, I decided to make 5 1:100 dilutions of the stock in order to produce a broad range of dilutions to test for plaque forming units:
    • Dilution 1: 1:100 50uL stock in 5mL T-broth
    • Dilution 2: 1:100 50uL dilution 1 in 5mL T-broth
    • Dilution 3: 1:100 50uL dilution 2 in 5mL T-broth
    • Dilution 4: 1:100 50uL dilution 3 in 5mL T-broth
    • Dilution 5: 1:100 50uL dilution 4 in 5mL T-broth
  • I decided to use 100uL from each of the last three dilutions just to see which dilution would be optimal for observing plaque forming units. I labeled my plates Blank #1, Blank #2, 100uL Dilution 3 #1, 100uL Dilution 3 #2, 100uL Dilution 4 #1, 100uL Dilution 4 #2, 100uL Dilution 5 #1, and 100uL Dilution 5 #2.
  • I could not use the Blank #1 plate because the top agar layer would not stick to the bottom agar. I did notice some water on the bottom agar before I poured the soft agar. This may be the cause of non-sticking top layer. Also, I may have disrupted this layer when I tilted to the plate to check to see if the top layer had solidified.
• I incubated both plaque groups at 2:15.
• I checked both plaque groups at 6:15 and found that there were plaques on all the plates except for the blanks and the dilution 4 and 5 groups for T7+ #2. For this reason, I allowed the T7+ #2 group to incubate a while longer in the hope that visible plaques would form on plates 100uL Dilution 4 #1-2 and 100uL Dilution 5 #1-2.
• I removed the 12 plates for T7+ #1 at 6:30 and found the following plaque counts:
  • Blank A: 0
  • Blank B: 0
  • Blank C: 0
  • 50uL A: 73
  • 50uL B: 80
  • 50uL C: 91
  • 100uL A: 151
  • 100uL B: 197
  • 100uL C: 178
  • 150uL A: 323
  • 150uL B: 345
  • 150uL C: 254
• The three plates 100uL A, 100uL B, and 150uL A each had streaks of plaque in the middle of the plate that made it difficult to tell plaques apart from each other. This may have been caused by water on the bottom agar before I poured the top agar.
• At 7:30, I removed the 8 plates for T7+ #2 and found the following:
  • Blank #1: I could not use this plate. The top agar did not solidify completely and formed a thin layer of agar that stayed separate from the bottom layer.
  • Blank #2: 0 plaques
  • 100uL Dilution 3 #1: Too many plaques were formed on this plate. Also, due to the extended amount of time that these plates were incubated, the plaques had grown very large and overlapped with each other, making it difficult to distinguish one from another.
  • 100uL Dilution 3 #2: Too many plaques were formed on this plate. Also, due to the extended amount of time that these plates were incubated, the plaques had grown very large and overlapped with each other, making it difficult to distinguish one from another.
  • 100uL Dilution 4: #1: 0
  • 100uL Dilution 4: #2: 0
  • 100uL Dilution 5: #1: 0
  • 100uL Dilution 5: #2: 0
• I suspect that problems that I encountered today (separated top layer of agar, streaks of plaque, no plaque) were due to the presence of water on many of the plates. For this reason, I removed the T-broth agar plates that I made last night from their plastic bag and placed them on the lab bench to dry overnight. Hopefully, this will help fix my problem.
• I made two more BL21 overnight cultures using the Making Overnight Suspensions protocol. I will use these cultures to make another attempt at finding the titer of the T7+ #2 stock.
• From the data for the T7+ #1 titer group, we can find an approximate titer for the T7+ #1 stock. I use the plaque counts from the 50uL group because I found no streaks of plaque in those groups and the numbers seem fairly close to each other:
  • Average for the 50uL of Dilution 4: 81.3 plaques
  • This translates to 1.6267e3 PFU/mL
  • I calculate the serial dilution backwards:
    • Dilution 4: (500uL)(1mL/1000uL)(Concentration 3) = (4.5mL)(1.626e3 PFU/mL)
      • Concentration 3 = 1.464e4 PFU/mL
    • Dilution 3: (500uL)(1mL/1000uL)(Concentration 2) = (4.5mL)(1.464e4 PFU/mL)
      • Concentration 2 = 1.3176e5 PFU/mL
    • Dilution 2: (500uL)(1mL/1000uL)(Concentration 1) = (4.5mL)(1.318e5 PFU/mL)
      • Concentration 1 = 1.186e6 PFU/mL
    • Dilution 1: (50uL)(1mL/1000uL)(Stock Concentration) = (5mL)(1.186e6 PFU/mL)
      • Stock Concentration = 1.19e8 PFU/mL

June 24, 2006

• I used the Making Plaques protocol to make 12 plates: Blank A, Blank B, Blank C, 50uL A, 50uL B, 50uL C, 100uL A, 100uL B, 100uL C, 150uL A, 150uL B, and 150uL C. I made 4 1:100 serial dilutions and used the final dilution as the phage stock for the plates. The dilutions were as follows:
  • Dilution 1: 1:100 50uL stock in 5mL T-broth
  • Dilution 2: 1:100 50uL dilution 1 in 5mL T-broth
  • Dilution 3: 1:100 50uL dilution 2 in 5mL T-broth
  • Dilution 4: 1:100 50uL dilution 3 in 5mL T-broth
• I touched the lip of the test tube for the 100uL B plate and I noticed bubbles in the soft agar of the 100uL C plate. I also dropped the lid of the 150uL B plate.
• I incubated all the plates at 12:30PM.
• I took out the plates at 4:30 and found the following plaque counts:
  • Blank A: 0 (still found water on the agar)
  • Blank B: 0
  • Blank C: 0
  • 50uL A: 7
  • 50uL B: 9
  • 50uL C: 9
  • 100uL A: 11
  • 100uL B: 22
  • 100uL C: 12
  • 150uL A: 34
  • 150uL B: 0
  • 150uL C: 15
• The bubbles in the agar of the 100uL C plate made it difficult to count plaques. The 100uL B plate seems to be inconsistent with the 100uL A and 100uL C plates which suggests that my touching the lip of the test tube holding the soft agar had an effect on either the amount of plaque or the amount of bacteria. Also, the presence of no plaque on the 150uL B plate suggests that dropping the lid of the plate affected either the bacterial growth or the infection.
• The plaque counts for the 50uL plates seem consistent, so we can use those numbers to calculate an approximate titer for the T7+ #2 stock:
  • Average plaque count for the 50uL plates: 8.33
  • This converts to 166.67 PFU/mL for Dilution 4
  • Dilution 4: (50uL)(1mL/1000uL)(Concentration 3) = (5mL)(166.67 PFU/mL)
    • Concentration 3 = 1.67e4 PFU/mL
  • Dilution 3: (50uL)(1mL/1000uL)(Concentration 2) = (5mL)(1.67e4 PFU/mL)
    • Concentration 2 = 1.67e6 PFU/mL
  • Dilution 2: (50uL)(1mL/1000uL)(Concentration 1) = (5mL)(1.67e6 PFU/mL)
    • Concentration 1 = 1.67e8 PFU/mL
  • Dilution 1: (50uL)(1mL/1000uL)(Stock Concentration) = (5mL)(1.67e8 PFU/mL)
    • Stock Concentration = 1.67e10 PFU/mL
• I placed all the plates in the 4*C fridge at 5:30PM and left 5 unused plates out to continue drying.

June 26, 2006

• Of the 5 plates left to dry, I bagged 4 of them and placed them in the 4*C fridge on my shelf. I may have contaminated one with my finger. I left this plate on the bench.
• I made two overnight cultures of IJ1126 cells using the Making Overnight Suspensions protocol. I used LB instead of T-broth in the protocol. I placed the plates in the 37*C incubation room at 6:40PM.
• Sri and I obtained two bottles of LB from the media room. One was used to make the overnights and is sitting on the lab bench. I placed the other bottle in the 37*C incubation room at 6:40PM.