User:Eric Ma/Notebook/MICB323 Lab Book/2009/03/03

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Part 1: Hybridization of Probe to DNA during Southern

  • Performed by MICB323 Instructors
  • Goal: To get DIG-labeled probe onto the membrane

Part 2: Detection of Probe using DIG Luminescent Detection System

  • Sunny did most of this part, while I did all of the 24-well plate and harvesting + Polyacrylamide Gel
  • After comparing Sunny's x-ray film to my own, we decided to use my film + gel image for the report.

Part 3: LPS +/- IFN-g Stimulation of Murine Macrophages

  • Macrophages were all prepared for us in DMEM media.
  • Sunny did "Student A" work - generating pooled samples for 24 hr stimulations.
  • I did "Student B" work - generating supernatants for analysis via ELISA.
  • I did not follow the protocol exactly. Rather than generating 3 time points until 4:30PM, I did 3 time points in 30 minute intervals. Hence, I expect to see less IFN-g stimulation compared to other groups'.
  • This is a table of the tubes that were generated:
Name Substance Added Time
EM PBS 0
EM PBS 0
EM LPS 30
EM LPS 30
EM LPS + IFNg 30
EM LPS + IFNg 30
EM LPS 60
EM LPS 60
EM LPS + IFNg 60
EM LPS + IFNg 60
EM LPS 90
EM LPS 90
EM LPS + IFNg 90
EM LPS + IFNg 90
EM IFNg 90
EM IFNg 90
EM LPS d1
EM LPS d1
EM LPS + IFNg d1
EM LPS + IFNg d1
EM IFNg d1
EM IFNg d1

Part 4: Pouring of Polyacrylamide Gel

  • Everything went smoothly after I botched up the gels the first time round. No problems for the 2nd try.