Protocols
Described here are the various techniques we used in the course of our project
Preparation of CaCl2 Competent Cells
- Dilute 400μl of overnight liquid culture into 20ml of broth with any necessary antibiotics to select for any plasmids already transformed into the cells
- Incubate at 37⁰C, shaking at 225rpm for 90 minutes
- Spin down for 2 minutes at 7000rpm at 4⁰C
- Discard supernatant, resuspend pellet in 10ml of 50 mM CaCl2, keep on ice
- Repeat centrifugation for 2 minutes at 7000rpm at 4⁰C
- Discard supernatant and the resuspend pellet in 1ml of 50 mM CaCl2, keep on ice
- CaCl2 competent cells can be kept on ice in the fridge overnight
Transformation of CaCl2 Competent Cells
- Add 1μl of plasmid DNA to 100μl of competent cells
- Incubate on ice for 20 minutes
- Heat shock at 42⁰C for 30 seconds or at 37⁰C for 5 minutes
- Keep on ice for 2 minutes
- Add 200μl of broth
- Incubate the cells at 37⁰C. The time varies with the antibiotic resistance gene:
Resistance Time Chloramphenicol 90 minutes Kanamycin 60 minutes Ampicillin 30 minutes - Spread 200μl of transformed cells on plates with required antibiotic to select for plasmids
- Incubate at 37°C overnight
Preparation of Electrocompetent Cells
- Inoculate 400ml L-broth with 4ml culture
- Incubate at 37⁰C, shaking at 250rpm until mid-log phase (OD600=0.5-0.7)
- Split culture into 2 x 200ml samples
- Chill on ice for 20 minutes
- Spin 4000g for 15 minutes at 4⁰C
- Re-suspend each in 200ml ice cold 10% glycerol
- Spin 4000g for 15 minutes at 4⁰C
- Re-suspend each in 100ml ice cold 10% glycerol
- Spin 4000g for 15 minutes at 4⁰C
- Re-suspend each in 10ml ice cold 10% glycerol
- Spin 4000g for 15 minutes at 4⁰C
- Re-suspend each in 500μl ice cold 10% glycerol
- Aliquot 60μl into small eppendorf tubes and store at -70⁰C
Transformation of Electrocompetent Cells
- Add 1μl of plasmid DNA to 30μl of competent cells
- Leave on ice for 20 minutes
- Transfer to pre-cooled electroporation cuvette
- An electrical pulse was delivered by a Biorad Micropulser and 1ml L-broth immediately added
- BCulture was then transferred to a 2ml nunc tube and incubated at 37⁰C for expression step (time varies dependent on antibiotic resistance gene in the plasmid that has just been transformed into the cells):
Resistance Time Chloramphenicol 90 minutes Kanamycin 60 minutes Ampicillin 30 minutes - Spread 100-200μl of transformed cells on dried L-agar plates with required antibiotic(s) to select for plasmid(s)
- Incubate plates at 37°C overnight
Restriction Digests
- A 20μl reaction typically contained:
- 2μl of buffer
- 4μl of miniprep (or 8μl G-Block) dependant on concentration of miniprep
- Make up to 20μl with ddH20
- 0.5-1.0μl of each restriction enzyme
- Vortex
- Incubate at 37⁰C for at least 60 minutes
- Chill on ice for 20 minutes
- Heat inactivate restriction enzymes
Miniprep
- Pipette 1mL of bacterial overnight culture into a microcentrifuge tube
- Centrifuge at 13,000 rpm for 1 min
- Discard the supernatant
- Repeat steps 1-3 two more times
- Add 250μl of P1 buffer and pipette up and down to resuspend the pellet
- Add 250μl of P2 buffer and invert Note: don’t allow this lysis reaction to proceed for more than 5 minutes
- Add 350μl of N3 buffer to neutralise the reaction and invert
- Centrifuge at 13,000 rpm for 10 minutes
- Transfer 800μl of supernatant into a column
- Centrifuge for 1 minute and discard flow-through
- Add 500μl of PB buffer and centrifuge for 1 minute
- Discard flow-through
- Add 750μl of PE buffer and centrifuge for 1 minute
- Discard flow-through
- Centrifuge again for 1 minute to get rid of any residual buffer
- Transfer the column to a microcentrifuge tube
- Add 50μl of EB buffer and let it stand for 1 minute
- Centrifuge for 1 minute
Making a gel
To make a 1L buffer:
To make a 1% agarose gel:
- Meaure 20mL of 50x TAE buffer
- Transfer to a 2L measuring cylinder
- Fill up to 1L with distilled water
To make a 1% agarose gel:
- Weigh out 1g of agarose powder
- Transfer to a microwave bottle
- Pour 100mL of your buffer into the bottle
- Microwave until clear
- Cool dwn to 55⁰C before pouring the gel
Shortbread
A good supply of shortbread is essential to any productive lab environment.
- Heat the oven to 190C/375F/Gas 5
- Beat the butter and the sugar together until smooth.
- Stir in the flour to get a smooth paste. Turn on to a work surface and gently roll out until the paste is 1cm/½in thick.
- Cut into rounds or fingers and place onto a baking tray. Sprinkle with caster sugar and chill in the fridge for 20 minutes.
- Bake in the oven for 15-20 minutes, or until pale golden-brown. Set aside to cool on a wire rack.