Difference between revisions of "Team:Glasgow/Protocols"

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<p>Here's the techniques we used blah blah blah"</p>
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<p>Described here are the various techniques we used in the course of our project</p>
 
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<li>Incubate at 37⁰C, shaking at 225rpm for 90 minutes</li>
 
<li>Incubate at 37⁰C, shaking at 225rpm for 90 minutes</li>
 
<li>Spin down for 2 minutes at 7000rpm at 4⁰C</li>
 
<li>Spin down for 2 minutes at 7000rpm at 4⁰C</li>
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<li>Discard supernatant, resuspend pellet in 10ml of 50 mM CaCl2, keep on ice</li>
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<li>Discard supernatant, resuspend pellet in 10ml of 50 mM CaCl<sub>2</sub>, keep on ice</li>
 
<li>Repeat centrifugation for 2 minutes at 7000rpm at 4⁰C</li>
 
<li>Repeat centrifugation for 2 minutes at 7000rpm at 4⁰C</li>
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<li>Discard supernatant and the resuspend pellet in 1ml of 50 mM CaCl2, keep on ice</li>
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<li>Discard supernatant and the resuspend pellet in 1ml of 50 mM CaCl<sub>2</sub>, keep on ice</li>
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<li>CaCl2 competent cells can be kept on ice in the fridge overnight</li>
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<li>CaCl<sub>2</sub> competent cells can be kept on ice in the fridge overnight</li>
 
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<li>1μl of plasmid DNA was added to 100μl of competent cells</li>
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<li>Add 1μl of plasmid DNA to 100μl of competent cells</li>
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<li>Samples were incubated on ice for 20 minutes</li>
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<li>Incubate on ice for 20 minutes</li>
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<li>Heat shock was cried out at 37⁰C for 5 minutes</li>
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<li>Heat shock at 42⁰C for 30 seconds <span class="i">or</span> at 37⁰C for 5 minutes</li>
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<li>Cells were kept on ice for 2 minutes</li>
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<li>Keep on ice for 2 minutes</li>
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<li>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):
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<li>Add 200μl of broth
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<li>Incubate the cells at 37⁰C. The time varies with the antibiotic resistance gene.
 
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<li>100-200μl of transformed cells was spread on dried L-agar plates with required antibiotic(s) to select for plasmid(s) </li>
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<li>Spread 200μl of transformed cells on plates with required antibiotic to select for plasmids</li>
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<li>Plates were incubated at 37°C overnight</li>
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<li>Incubate at 37°C overnight</li>
 
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Revision as of 10:00, 2 August 2016

Glasgow iGEM 2016
Protocols

Described here are the various techniques we used in the course of our project

Preparation of CaCl2 Competent Cells
  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
Transformation of CaCl2 Competent Cells
  1. Add 1μl of plasmid DNA to 100μl of competent cells
  2. Incubate on ice for 20 minutes
  3. Heat shock at 42⁰C for 30 seconds or at 37⁰C for 5 minutes
  4. Keep on ice for 2 minutes
  5. Add 200μl of broth
  6. Incubate the cells at 37⁰C. The time varies with the antibiotic resistance gene.
    Resistance Time
    Chloramphenicol 90 minutes
    Kanamycin 60 minutes
    Ampicillin 30 minutes
  7. Spread 200μl of transformed cells on plates with required antibiotic to select for plasmids
  8. Incubate at 37°C overnight
Preparation of Electrocompetent Cells
  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
Transformation of Electrocompetent Cells
  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
Restriction Digests
  1. 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
  2. Vortex
  3. Incubate at 37⁰C for at least 60 minutes
  4. Chill on ice for 20 minutes
  5. Heat inactivate restriction enzymes
Miniprep
  1. Pipette 1mL of bacterial overnight culture into a microcentrifuge tube
  2. Centrifuge at 13,000 rpm for 1 min
  3. Discard the supernatant
  4. Repeat steps 1-3 two more times
  5. Add 250μl of P1 buffer and pipette up and down to resuspend the pellet
  6. Add 250μl of P2 buffer and invert Note: don’t allow this lysis reaction to proceed for more than 5 minutes
  7. Add 350μl of N3 buffer to neutralise the reaction and invert
  8. Centrifuge at 13,000 rpm for 10 minutes
  9. Transfer 800μl of supernatant into a column
  10. Centrifuge for 1 minute and discard flow-through
  11. Add 500μl of PB buffer and centrifuge for 1 minute
  12. Discard flow-through
  13. Add 750μl of PE buffer and centrifuge for 1 minute
  14. Discard flow-through
  15. Centrifuge again for 1 minute to get rid of any residual buffer
  16. Transfer the column to a microcentrifuge tube
  17. Add 50μl of EB buffer and let it stand for 1 minute
  18. Centrifuge for 1 minute
Shortbread
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.