Items needed
1 - DNA Plasmid
1- Plate of E. coli K12 derived DH5a or DH10B or BL21 bacteria
2 - LB Agar plates
5mL Transformation buffer (10% PEG 8000, 5% DMSO, 25mM CaCl2)
18g LB Amp Agar or LB Kan Agar
20 plates
250mL glass jar
10 inoculation loops
50 - 1.5mL microfuge tubes
10 Dropper pipettes
Restreak the DH5a, DH10B or BL21 cells every 1-2 months. Fresh cells will provide better transformation efficiency.
18g of LB Agar should pour ~20 plates from 500mL
If you have a centrifuge start at (1) otherwise start at (8)
- Grow up 5mL overnight culture in LB and the appropriate antibiotic
- Spin down at ~3000rpm
- Decant/dump out LB supernatant
- Resuspend cells in 500uL to 1mL of Bacterial Transformation Buffer
- Spin down cells at ~3000RPM
- Decant/dump out transformation buffer supernatant
- Add 100uL transformation buffer to the cell pellet and gently resuspend them
- If you do not have access to a centrifuge using an inoculation loop scrape enough bacteria to almost fill the loop and mix it into 100uL transformation buffer.
- Add 500ng+ of plasmid to the microfuge tube containing the transformation buffer and place at 4C or in the fridge for 30 minutes.
- “Heat Shock” the bacteria by placing them in water that is ~42C for 30 seconds.
- Then transfer bacteria to a microcentrifuge tube of recovery media (LB broth) and let sit at 37C(room temperature is fine if you don’t have a 37C incubator) for 1-3 hours (in this step the bacteria that have accepted the DNA will produce antibiotic resistant proteins for selection and replicate hopefully creating more bacteria with the plasmid)
- Take 100uL of your transformation and put it on a LB agar plate with the correct antibiotic to select for your plasmid
- Incubate overnight at 37C or ~24 hour at room temperature(RT)(if using the pVIB.pJE202 bioluminescent plasmid incubate only at RT).