Bacmid Transformation and Bacterial Culture
📅 8.18
Operator: Miao Lexing, Zhang Yichi
- Prepared 200ml LB medium and sterilized it
- Diluted CM plasmid to 400 ng/μl, added 1μl to competent cells, and let stand for 30 minutes
- Heat shock at 42°C for 90 seconds, added 1ml antibiotic-free LB medium for recovery (4 hours, 37°C shaker at 180 rpm)
- Stored remaining plasmid at -20°C
- Prepared culture plates: 200ml LB medium, added 200μl Kanamycin, Gentamicin, Tetracycline, and Bluo-gal, added 33μl IPTG. Poured into ten culture dishes
- Diluted one group of bacterial solution to 10x and 100x. Took 200μl each of 1x, 10x, and 100x bacterial solutions and spread evenly onto triple-antibiotic plates. Cultured in shaker for two days
📅 8.25
Operators: Yang Ruiming, Miao Lexing
- Checked culture results from 8.18, selected two larger white colonies, and froze them
- Performed transformation on the remaining three plasmids (MS, SC, MCS): Diluted each to 400ng/μl, added 1μl to competent cells, stood for 30min
- Heat shock at 42°C for 90 seconds, added 1ml antibiotic-free LB medium for recovery (4 hours, 37°C shaker at 180 rpm)
- Stored remaining plasmids at -20°C
- For the three groups of bacterial solutions, diluted 200μl to 10x for each. Spread both original and diluted solutions onto triple-antibiotic plates. Cultured for two days
📅 8.28
Operators: Zhang Jingtian, Miao Lexing
- The 8.25 cultures showed no growth, suspected issue with competent cells
- Repeated transformation with new competent cells, operation identical to 8.25
- Prepared 200ml triple-antibiotic LB during recovery period
📅 8.31
Operators: Miao Lexing, Zhang Jingtian, Yang Ruiming
- Prepared 4 groups: 6ml medium * 4, with antibiotics (Kanamycin, Tetracycline, Gentamicin (1000x))
- Picked 2 SC white colonies and 2 CM white colonies
- Shaker culture for 1 day
📅 9.1
Operators: Miao Lexing, Zhang Jingtian, Yang Ruiming
- Repeated transformation of SM and MCS plasmids, operation same as above
- Performed viral plasmid extraction
- Measured concentration, sufficient for next step experiments
Virus Transfection and Cell Culture
📅 9.14
Electrophoresis
- Order from left to the right: SC1 SC2 MS1 marker MS2 CM1 CM2
📅 9.15
Operator: Yang Ruiming
- Took 14 tubes, added 100μl insect cell medium to each
- Added 10μl transfection reagent to 7 tubes, mixed
- Added virus plasmid (1μg each) to the other 7 tubes, mixed gently by flicking
- Added 200μl medium containing transfection reagent to these 7 tubes
- Took the 6-well plate from the previous day, removed supernatant, added 800μl medium containing the plasmid mixture, mixed, and added to the 6-well plate
- Cultured for 5 days
📅 9.20
Virus Harvesting
- Harvested supernatant from the 6-well plate cultured on the 15th
- Took two new 6-well plates, added 0.5mL cell culture medium (containing cells) to each well
- Prepared three culture dishes, added 2mL of remaining cells to each dish
- Added 8mL medium to each culture dish
- Added all harvested virus to the aforementioned 6-well plates, with virus type corresponding to each plate
- Cultured at 28°C for 3 days
📅 9.27
Western Blotting - Primary Antibody
- Suspended cells in 1mL buffer
- Added 20mL protease inhibitor, then lysed with ultrasound for 30s
- Mixed 80μl protein + 20μl dye, metal bath at 95°C for 10 min
- Loaded gel (left to right): Marker (5μl) + seven samples (10μl each) + Marker (2.5μl). Electrophoresis at 120V for 50 min, transfer at 400mA for 2h
- Added skim milk for blocking, 1h
- Added skim milk containing primary antibody, placed in 4°C refrigerator for 12-16 hours
Cell Lab
Key Activities involving Cells
Virus transfection and amplification in insect cells (9.15, 9.20)
Cell collection and lysis for Western Blotting (9.27)
Cell Freezing & Viability Assessment: Preparation of freezing medium, cell detachment, dividing into replicate tubes, and Trypan Blue staining to record viability for the purpose of comparing cell viability before and after the freeze-thaw process (9.29)
Cell Thawing & Viability Assessment: Thawing of cells, warm water renaturation, and statistical viability assessment for the purpose of comparing cell viability before and after the freeze-thaw process (9.30)
Subsequent culture of thawed cells (10.2)
📅 9.29
Operators: Yang Ruiming, Zhang Jingtian, Miao Lexing
- Prepared cell freezing medium
- Detached cells, divided cells corresponding to each plasmid into three tubes as replicates
- Performed Trypan Blue staining to record viability for the purpose of comparing cell viability before and after the freeze-thaw process
📅 9.30
Western Blotting - Secondary Antibody
- Membrane regeneration solution, shaker for 15 min
- Prepared 50ml 5% skim milk
- Took cells, warm water renaturation, statistically viability for the purpose of comparing cell viability before and after the freeze-thaw process
📅 10.2
First Experiment Cycle Completion
- The first experiment cycle completed
- Transferred the cells resuscitated the previous day to culture dishes, added 5ml medium, and continued cultivation