3rd Attempt at Electroporating pSPIN-BAC Cells with DiNV DNA for DiNV-BAC Integration
Plan on doing 10 plates and screening ~100 colonies per plate, so like 1000 colonies checked from this attempt…
Prepping Plates
- Making 10LB plates for first streaking
- Making all plates with chloramphenicol resistance, also adding kanamycin to 2 of them
- Made a diluted solution of chlor:
- 150ul 100% ethanol
- 150ul stock chlor
- Spread 20ul of chlor solution on 10 LB plates, each with a new glass spreader from a pastur pipette
- For 2 plates added an additional 20ul of stock kanamycin
- Let plates sit on bench to warm
Electroporation
- Prepped 2 tubes of 975 SOC buffer
- Thawed 1 tube of electrocompetent pSPIN-BAC cells
- Using 26-exo sample
- Brought up to Chandler lab:
- ice with bacteria and DNA
- pipettes and tips
- bacterial tip waste
- tape
- 20 1.5mL tubes
- tubes with SOC buffer
- Placed SOC in 30C incubator to warm
- Placed cuvette on ice to chill
- Electroporation sample (just 1):
- Added 25ul cells to 1.5mL tube on ice
- Added 2.5ul of 26-exo DNA
- Mixed and transferred to cuvette on ice
- Electroporated EC2 settings
- Added 975ul warmed SOC and mixed
- Transferred solution to 1.5mL tube
- Incubated tube at 30C for 1 hour shaking
Wash and Plate
- Warmed LB to room temp
- After incubation, took tube to 4012 and centrifuged 3 min at 3,000rpm
- Removed supernatant
- Resuspended pellet in 100ul of LB
- Spread 10ul of bacteria solution on each of the 10 plates made earlier with a sterile loop spreader
- Plates 1 and 2 had the chlor and kan resistance
- Placed plates in the 30C incubator to grow
At low temp it takes a while to grow, so they went ~1.5 days and were put in the fridge at 9pm the next day