User:Eric Ma/Notebook/MICB323 Lab Book/2009/01/13

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Lab 1: PCR, Modification of PCR Component, and Phage T4 Growth Curve

Part 1: PCR

  • Protocol as specified in MICB323 Lab Manual pages 8-9
  • Partner: Sunny
  • See this file for experimental values used.
  • 200µL Thin-Walled Tube Numbers:
    • ES #1: Primer 1 + DNA 1
    • ES #2: Primer 1 + DNA 2
    • ES #3: Primer 2 + DNA 1
    • ES #4: Primer 2 + DNA 2
  • Tubes put into ice.

Part 2: Modification of PCR Component

  • Protocol as specified in MICB323 Lab Manual page 11
  • See this file for experimental values used.
  • Variable chosen to vary: KCl concentration
  • 200µL Thin-Walled Tube Numbers:
    • EM #1: 10X Buffer contains 0.25M KCl
    • EM #2: 10X Buffer contains 0 M KCl
    • EM #3: 10X Buffer contains 0.125M KCl (0.5X the concentration of original)
    • EM #4: 10X Buffer contains 1M KCl (4X the concentration of original)
  • Tubes placed into ice

Part 3: Phage T4 Growth Curve

  • Protocol followed as shown in pages 16 to 19 in the MICB323 Lab Manual
  • Changes Made:
    • 15 minute timepoint was changed to 16 minutes due to unforeseen delay.
    • Other timepoints were followed exactly.
  • Group Division:
    • Person A: Frank
    • Person B: Jeffrey
    • Person C: Sunny
    • Person D: Eric
  • Other Data:
    • Temperature of incubation water bath: 34ºC
    • Temperature of shaking water bath: 35ºC
  • Some Timepoints:
    • Pre-Incubation
      • Time E. coli and phage were separately incubated in water bath: 2.57PM
      • Time E. coli and phage were taken out of water bath: 3.02PM
      • Phage and E. coli were mixed at this point
    • T=-5min
      • Time E. coli and phage were incubated together in water bath: 3.04PM
      • Time E. coli and phage were taken out of water bath: 3.09PM
    • T=0
      • Time phage and E. coli were put into flask: 3.12PM
      • Time phage and E. coli were put into shaking water bath: 3.14PM (this is T=0 min)
  • Things to note:
    • As there was not enough E. coli culture left in the tubes shared at my bench, I had to go to look for fresh cultures and used cultures for use in the plating steps. I'm not sure how this will affect the results, but it certainly would be interesting to see if there were any effects or not on the experiments.

Summary

  1. First PCR prep went well, no signs of any major problems.
  2. Second PCR prep also went well, no signs of major problems.
  3. I may need to take note of the fact that many cultures were used for the bacteriophage experiments - that may become significant when doing the plate counts.
    1. Will the bacteria grow well?
    2. Are they all from the same stock source?
    3. Will there be any contamination?



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