Protocols




April-May


  • Training: live cell imaging, trypan blue cell counting, fluorescence microscopy, virus packaging, etc.
  • Preparation for E. coli dosage tests: transformation and midi prep


Week 1 (April 17-23)

  1. Training live cell imaging with two-photon excitation microscopy/ confocal microscopy, using eGFP, and mCherry transfected stem cells


Week 2 (May 1-7)

  1. Preparing tips, cells, and medium
  2. Training with a fluorescence microscope
  3. Training cell counting with trypan blue


Week 3 (May 8-14)

  1. Transformation of GFP control into Top10 cells


Week 4 (May 15-21)

  1. Midi prep to extract GFP from Top10 cells
  2. Virus packaging of GFP to HEK cells


June


  • Top10 dosage tests with UV: finding suitable concentrations and intensities of chemicals and UV to induce DNA damage
  • Continue preparation for E. coli dosage tests: transformation, midi prep, and preparing bacterial stock
  • Preparation for FRET biosensor: virus packaging and extraction


Week 5 (May 29-June 4)

  1. Top10 dosage tests with UV


Week 6 (June 5-11)

  1. Transformation of RecA-EGFP (K3 and K6)
  2. Midi for RecA (K6 is successful)


Week 7 (June 19-25)

  1. Transformation for K3-EGFP-hha, K6-EGFP-hha, K3-hha
  2. Midi for K3-EGFP-hha, K3-hha
  3. Pass HEK cells & E. coli stocking

July


  • Preparation for FRET biosensor: virus packaging, finding a suitable cell line that can express our viruses
  • E. coli dosage tests with UV and H2O2: the plate reader data was not conclusive, so we decided to use cell imaging for further validation


Week 8 (June 26-July 2)

  1. Virus packaging and extraction(mCherry eGFP eYFP eCFP)
  2. K6-EGFP-hha transformation and stocking
  3. Pass cell (HEK293 and B16F10)


Week 9 (July 3-9)

  1. Infect HEK293 and B16F10
  2. E.coli UV and H2O2 test
  3. Thaw cells (HEK293 and B16F10)


Week 10 (July 10-16)

  1. E.coli UV and H2O2 test, with cell imaging
  2. Pass cell (HEK293 and B16F10)
  3. Virus packaging


Week 11 (July 17-23)

  1. E.coli UV and H2O2 test, with cell imaging
  2. Pass cell (HEK293 and B16F10)


Week 12 (July 24-30)

  1. E.coli UV and H2O2 test, with cell imaging
  2. Pass cell (HEK293 and B16F10)
  3. Midi for CFP, YFP, mCherry control




August


  • E. coli test with UV, H2O2, nalidixic acid, and aspartame: Using plate readers and cell imaging to quantify fluorescence
  • Preparation for FRET biosensor: virus packaging and infection, using fluorescent microscopy to find a suitable cell line, continue selecting cells
  • Contamination of mammalian cells: use HBSS to wash cells


Week 13 (July 31-August 6)

  1. E. coli test with UV, H2O2, nalidixic acid, and aspartame, with cell imaging
  2. Infect and select B16F10 and HEK293


Week 14 (August 7-13)

  1. E. coli test with UV, H2O2, nalidixic acid, and aspartame, with cell imaging
  2. Pass cell (A2780, OVC, B16F10, HEK293)
  3. Select B16F10 and HEK293 and photograph with fluorescence microscope
  4. Pack virus


Week 15 (August 14-20)

  1. Pass, infect and select OVC, A2780
  2. Pack virus
  3. E. coli test with UV, H2O2, nalidixic acid, and aspartame, with cell imaging
  4. Seed mCherry and YFP into 6 well plate


Week 16 (August 21-27)

  1. Infect mCherry and YFP with GFP and CFP respectively
  2. Transformation and midi for DVPR, VSVG
  3. Bacteria identified, only eGFP-mCherry is viable
  4. Remake medium and use HBSS to wash cells




September-October


  • Contamination of mammalian cells: repack viruses because old viruses are contaminated
  • Preparation for FRET biosensor: infecting and passing selected cells to confocal dish
  • Observing FRET: Using confocal microscopy find the optimal intensity of UV, and observe the congregation of fluorescence before and after UV treatment, quantifying FRET
  • E. coli with different RBS tests with UV, H2O2, nalidixic acid, and aspartame


Week 17 (August 28-September 3)

  1. Pass cell (HEK293 and A2780)
  2. Repack viruses


Week 18 (September 4-10)

  1. Infect A2780 and HEK293 (mCherry, eGFP, eYFP, eCFP, mCherry-eGFP, eYFP-eCFP)
  2. Pass YFP and mCherry (HEK293) to 6 well plate
  3. Continue to wash cells with HBSS


Week 19 (September 11-17)

  1. Pass YFP, GFP, mCherry, and GFP-mCherry (HEK293) to confocal dishes and UV test
  2. Infect and select CFP-YFP (HEK293)
  3. TUNEL assay on HEK cells


Week 20 (September 18-24)

  1. Infect CFP and CFP-YFP (HEK293)
  2. Transformation and midi prep for E. coli with different RBS (B0032, strong)
  3. E. coli test of new RBSs with UV, H2O2, nalidixic acid, and aspartame
  4. Pass GFP, GFP+mCherry (mCherry double infected), mCherry (double infected) to confocal dishes


Week 21 (September 25-October 1)

  1. Pack CFP and YFP virus
  2. Thaw HEK293 cells, re-infect with CFP and YFP, then select
  3. UV test on cells in confocal dishes from week 20
  4. Pass CFP, CFP+YFP, GFP to confocal dish and do UV test


Week 22 (October 2-8)

  1. E. coli test of new RBSs with UV, H2O2, nalidixic acid, and aspartame
  2. mCherry and GFP-mCherry UV test