Difference between revisions of "Team:Glasgow/Protocols"

1. Dilute 400μl of overnight liquid culture into 20ml of broth with any necessary antibiotics to select for any plasmids already transformed into the cells
2. Incubate at 37⁰C, shaking at 225rpm for 90 minutes
3. Spin down for 2 minutes at 7000rpm at 4⁰C
4. Discard supernatant, resuspend pellet in 10ml of 50 mM CaCl2, keep on ice
5. Repeat centrifugation for 2 minutes at 7000rpm at 4⁰C
6. Discard supernatant and the resuspend pellet in 1ml of 50 mM CaCl2, keep on ice
7. CaCl2 competent cells can be kept on ice in the fridge overnight

1. 1μl of plasmid DNA was added to 100μl of competent cells
2. Samples were incubated on ice for 20 minutes
3. Heat shock was cried out at 37⁰C for 5 minutes
4. Cells were kept on ice for 2 minutes
5. 200μl of broth was added and the cells 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

6. 100-200μl of transformed cells was spread on dried L-agar plates with required antibiotic(s) to select for plasmid(s)
7. Plates were incubated at 37°C overnight

1. Inoculate 400ml L-broth with 4ml culture
2. Incubate at 37⁰C, shaking at 250rpm until mid-log phase (OD600=0.5-0.7)
3. Split culture into 2 x 200ml samples
4. Chill on ice for 20 minutes
5. Spin 4000g for 15 minutes at 4⁰C
6. Re-suspend each in 200ml ice cold 10% glycerol
7. Spin 4000g for 15 minutes at 4⁰C
8. Re-suspend each in 100ml ice cold 10% glycerol
9. Spin 4000g for 15 minutes at 4⁰C
10. Re-suspend each in 10ml ice cold 10% glycerol
11. Spin 4000g for 15 minutes at 4⁰C
12. Re-suspend each in 500μl ice cold 10% glycerol
13. Aliquot 60μl into small eppendorf tubes and store at -70⁰C

1. Add 1μl of plasmid DNA to 30μl of competent cells
2. Leave on ice for 20 minutes
3. Transfer to pre-cooled electroporation cuvette
4. An electrical pulse was delivered by a Biorad Micropulser and 1ml L-broth immediately added
5. 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

6. Spread 100-200μl of transformed cells on dried L-agar plates with required antibiotic(s) to select for plasmid(s)
7. Incubate plates at 37°C overnight

A good supply of shortbread is essential to any productive lab environment.

1. Heat the oven to 190C/375F/Gas 5
2. Beat the butter and the sugar together until smooth.
3. 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.
4. Cut into rounds or fingers and place onto a baking tray. Sprinkle with caster sugar and chill in the fridge for 20 minutes.
5. Bake in the oven for 15-20 minutes, or until pale golden-brown. Set aside to cool on a wire rack.