Cycle 1
Wet lab workflow in SSC
- Design the Plasmid FLG ✅
- Clone the plasmid in E.coli DH5alpha✅
- Single bacterial colony inoculation✅
- Plasmid isolation followed by NanoDrop✅
- Confirmation of clone by restriction digestion✅
- Running gel electrophoresis✅
- Sub cloning the plasmid into E.coli BL21 ✅
- Confirmation of clone by colony PCR ✅
- Single colony inoculation✅
- IPTG induction and protein expression✅
- Running the SDS protein gel✅
- Protein extraction✅
- Plotting bacterial growth curve against the protein expression.✅
Workshop in CUHK
- SCRATCH assay using heat shock protein and collagen✅
Wet lab in HKU
- qPCR for Carbonic anhydrase II (CA2), IL-24, ribosomal protein L38 (RPL38) and FLG ✅
Cycle 2
- Design the Plasmid FLG✅
- Clone the plasmid in E.coli DH5alpha✅
- Colony PCR for the confirmation of clone😞
- Gel electrophoresis
- Plasmid isolation
- Clone the plasmid into BL21
- Colony PCR for the confirmation of clone
- Gel electrophoresis
Day Count | Date | Work | Link to protocol | Notes |
Day 1 | 13/5 | Media preparation | ||
Day 2 | 14/5 | Hydration of plasmid | ||
Day 3 | 15/5 | Transformation and plating | ||
Day 4 | 16/5 | Review transformation results | Fail, no colonies observed | |
Day 5 | 19/5 | Transformation | Modification: 45 seconds for heat shock | |
Day 6 | 20/5 | Review transformation results | Success, although one negative control is contaminated | |
Day 6 | 20/5 | Growing single bacteria colony | ||
Day 7 | 21/5 | Plasmid isolation | No.1,3,4 were dropped and spilled | |
Day 7 | 21/5 | Nanodrop | All the sample concentration are very low | |
Day 8 | 26/5 | Inoculation | ||
Day 9 | 27/5 | Plasmid isolation and nanodrop | Highest concentration: 113ng/uL | |
Day 10 | 28/5 | Restriction digestion and gel electrophoresis | ||
Day 11 | 25/6 | Media | ||
Day 12 | 26/6 | Workshop at CUHK | Seeded cells and did scratch assay | |
Day 13 | 27/6 | Workshop at CUHK | ||
Day 14 | 3/7 | Transformation | ||
Day 15 | 10/7 | ColonyPCR preparation | ||
Day 16 | 14/7 | ColonyPCR | ||
Day 17 | 15/7 | Gel electrophoresis and colonyPCR | 15/7 Day17 Gel electrophoresis and single colony inoculation | No bands observed, failed |
Day 18 | 16/7 | Gel electrophoresis | ||
Day 19 | 17/7 | IPTG induction | ||
Day 20 | 18/7 | Cell harvesting | ||
Day 21 | 22/7 | Protein extraction | ||
Day 22 | 23/7 | colonyPCR and cell harvesting | ||
Day 23 | 24/7 | SDS-PAGE | ||
Day 24 | 25/7 | Sample preparation for SDS PAGE for 0.5mM | ||
Day 25 | 28/7 | SDS-PAGE for 0.5mM | ||
Day 26 | 30/7 | Sample preparation for 1mM SDS-PAGE | ||
Day 27 | 31/7 | Destaining of SDS PAGE 1mM and Bacteria growth curve | Dont know how to use the spectrophotometer | |
Day 28 | 1/8 | Bacteria growth curve at37°C | ||
Day 29 | 4/8 | Bacteria growth curve at 37°C | ||
Day 30 | 6/8 | Bacteria growth curve at 37°C | ||
Day 31 | 7/8 | Bacteria growth curve at 37°C and Cell lysate | ||
Day 32 | 8/8 | Bacteria growth curve at 37°C | ||
Day 33 | 11/8 | preparing primary culture, FLG purification at RT, cell lysate | ||
Day 34 | 12/8 | IPTG expression at 20°C, bacterial growth curve, FLG purification at 37°C | ||
Day 35 | 13/8 | IPTG expression at 20°C, preparing FLG at 37°C for SDS-PAGE | ||
Day 36 | 15/8 | |||
Day 37 | 18/8 | SDS-PAGE for total protein at 20°C | ||
Day 38 | 19/8 | Destaining of SDS-PAGE gel | ||
Day 39 | 20/8 | Dry lab work | ||
Day 40 | 21/8 | Dry lab work | ||
Day 41 | 25/8 | HKU lab Seeding cells | ||
Day 42 | 27/8 | HKU lab preparing stock, working concentration and wells | ||
Day 43 | 28/8 | HKU lab harvesting cells at T24 | ||
Day 44 | 29/8 | HKU lab harvesting cells at T48 | ||
Day 45 | 2/9 | HKU lab Extracting RNA T1 | ||
Day 46 | 3/9 | CYCLE 2 START Media preparation 200ml agar, 10 plates | ||
Day 47 | 4/9 | Transformation and plating | ||
Day 48 | 5/9 | HKU lab Extracting RNA T24 and T48 | ||
Day 49 | 9/9 | HKU lab reverse transcription of all samples | ||
Day 50 | 10/9 | Colony PCR for idT-pET-FLG-RFP | ||
Day 51 | 11/9 | Gel electrophoresis | ||
Day 52 | 16/9 | Single colony inoculation | ||
Day 53 | 17/9 | Plasmid isolation + nanodrop | ||
Day 54 | 18/9 | HKU qPCR | ||
Day 55 | 19/9 | RD for confirmation of clone | Failed again ah;( |