Preparation of calcium competent Escherichia coli and heat-shock transformation

(References Chang A et al. (2017) Preparation of calcium competent Escherichia coli and heat-shock transformation. JEMI Methods 1:22–25)

>MATERIALS AND EQUIPMENT
Materials:
1. Competent cell preparation:
2. 1mL of overnight Escherichia coli (E.coli) culture
3. 100mL of 0.1M CaCl2 (ice cold)
4. 20mL of 0.1M CaCl2 with 15% glycerolsolution (ice cold)
5. 100mL of fresh lysogeny broth (LB)media

Heat-shock transformation:
1. 1pg -100ng plasmid DNA (1-5uL)
2. 1mL of pre-warmed LB media or SOC media at 37℃
3. LB agar plates (with appropriate reagent for selective or screening)
4. Ice

Equipment:
1.    37°C shaking incubator
2.    42°C water bath
3.    Spectrophotometer

METHODS
A. CaCl2 Buffers Preparation
1M CaCl2 (stock solution, 10x working concentration)
• Weigh out 11.1g of anhydrous CaCl2
• Add to 80mL of dH2O
• Mix solution until CaCl2 is fully dissolved
• Top up to 100mL
• Filter sterilize through a 0.22μm pore

0.1M CaCl2 (working solution)
• Add 10mL of 1M CaCl2 to 90mL of dH2O for a 1:10 dilution
• Filter sterilize through a 0.22μm pore

0.1M CaCl2 + 15% glycerol (working solution)
• Mix 6mL 1M CaCl2 with 9mL sterile glycerol and 45mL dH2O

B. Overnight Culture(s)
• Inoculate 1mL of LB with E. coli
• Place in shaking incubator at 37°C and 200rpm
• Incubate for 12-16 hours

C. Generation of Competent Cells (CaCl2 wash)

Subculturing overnight culture:
• Add 1mL of overnight culture to 99mL of fresh LB (1:100 dilution, no antibiotics)
• Shake incubate at 37°C and 200rpm for 3-4 hours or until OD reaches 0.4

CaCl2 wash:
• Ensure that all reagents (CaCl2 solutions,Oakridge tubes, centrifuge) are ice-cold or at 4°C
• Separate culture into multiple Oakridge tubes
• Place on ice for 20 minutes
• Centrifuge at 4°C at 4000rpm for 10 minutes
• Discard the supernatant by tipping tubes over a discard bin and then aspirating any remaining media
• Resuspend each pellet with 20mL ice-cold 0.1M CaCl2, incubate on ice for 30 minutes
• Centrifuge at 4°C at 4000rpm for 10 minutes
• Discard the supernatant and combine pellets by resuspending in 5mL ice-cold 0.1M CaCl2 with 15% glycerol
• Use for downstream transformation or store in -80°C freezer
D. Heat-shock transformation
Heat-shock:
• Thaw competent cells on ice
• Add 1-5μl (10pg-100ng) of plasmid (do not exceed 5μL for a 50μL cell aliquot)
• Incubate on ice for 30 minutes
• Heat-shock by placing in 42°C water bath for exactly 30 seconds
• Place cells on ice for 2 minutes
• Add 1mL pre-warmed LB or SOC medium
• Shake incubate 37oC, 200rpm, 1 hour for outgrowth

Plating and incubation:
• Spread plate 1:10 and 1:100 dilutions of the outgrowth cultures on warm selective and/or screening plates (e.g. Ampicillin and/or X-gal if required)
• Incubate at 37°C for 12-16 hours

Plate observations:
• Inspect plates for isolated colonies

ANTICIPATED RESULTS AND CONTROLS
Anticipated results:
• No colonies appear on the plate.
• A countable number of colonies (30-300 colonies) appear on the plate.
• Many colonies that are too numerous to count appear on the plate.
Controls:
• Use a DNA preparation that has been shown to give transformants in previous experiments to act as a positive control.
• Perform another transformation to which plasmid DNA is not added to act as a negative control.
• Transform 1ng of target plasmid to check competent cell viability, calculate transformation efficiency, and verify the antibiotic resistance of the plasmid.