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Lab Supplies=Pelling:Lab Supplies
Lab Safety and MSDS's=Pelling:Lab Safety
Preparing DH5α Chemically Competent Cells
- 2M CaCl2 Stock Solution (22.198 grams CaCl2, Anhydrous into 100mL sterile dH2O and filter sterilize).
- LB Medium
- LB-Agar Plates (no antibiotics)
- Ice cold dH2O
- Autoclave 6 x 50mL tubes
- Autoclave 500mL Erlenmeyer Flask
- Autoclave 15 x 1.5mL microcentrifuge tubes
CaCl2 Competent Cell Stock Protocol
- Day 1 Afternoon: Streak cells from a frozen stock and grow overnight.
- Day 2 Afternoon: Pick single colony and grow in 5mL LB media overnight (300rpm).
- Day 3 Morning:
- Chill the centrifuge to 4°C and continue to Steps 2 and 3. DO NOT start Step 4 before centrifuge reaches 4°C.
- Make 20mL of 0.5M CaCl2 in ice cold sterile dH2O in a sterile 50mL tube. Leave on ice in the fridge. (5mL 2M CaCl2 + 15mL dH2O).
- Make 6mL sterile freezing media (0.1M CaCl2 / 15% Glycerol) in a sterile 50mL tube. Leave on ice in the fridge. (0.3mL 2M CaCl2 + 0.9mL 100% Glycerol + 4.8mL dH2O).
- Transfer into a 500mL Erlenmeyer Flask with 195mL LB media and grow cells until an OD of 0.5-0.6 A600 is achieved.
- Split cells into 4 x 50mL sterile tubes and put on ice and keep chilled at 0°C for 15min.
- Keep everything from this point onwards at 4°C or lower.
- Spin cells at 4000rpm for 10 min at 4°C.
- Remove supernatant, collect and re-suspend cells with 15mL ice-cold 0.5M CaCl2 by gentle pipetting (DO NOT VORTEX). Leave cells on ice for 15min.
- Spin again at 4000rpm for 10 min at 4°C.
- Re-suspend in 6mL sterile, ice-cold 0.1M CaCl2 /15% glycerol (DO NOT VORTEX).
- Optional: Leave on ice 4 to 21 hours (DH5α can be left overnight).
- Freeze 500μL aliquots of cells in sterile and labeled microcentrifuge tubes.
Transforming DH5α Chemically Competent Cells
- Thaw an aliquot of chemically competent bacteria on ice for 15min.
- Add 1-2μl (up to 10μl) of plasmid DNA (ng amounts) to 50μl of bacteria in a sterile tube. Want to use small amounts of DNA as large volumes will dilute bacteria buffer and reduce efficiency. Keep previously extracted DNA samples in case anything goes wrong.
- Incubate tubes on ice for 30min.
- Heat shock cells by placing tube in water at 42°C for exactly 45sec.
- Immediately transfer tubes to ice for 2min.
- Add cold 250μl of LB or SOC media. SOC better if LB doesn’t work.
- Cap the tube tightly and put the tubes horizontally in the shaker (tape them down) and shake at 37°C for 60-90mins.
- If necessary make dilutions of the bacteria in (1:10, 1:2 or 1:1) in LB in new tubes.
- Place 100μl of bacteria in center of an appropriate LB plate and spread evenly over whole surface with bent Pasteur pipette or any other loop, tip or spreader.
- Most strains require 12-18 hours to form colonies. Do not incubate for excessive times as satellite colonies will form (E.coli form small colonies). Plates/colonies can be stored for up to a week (with parafilm) at 4°C if not to be used immediately.
- Place any unused chemically competent bacteria back in the -80°C freezer and mark top with a black 'X' to denote that it has been thawed at least once.