Team:Imperial/Protocols

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Imperial iGEM 2014

Protocols

Here you can find the protocols we used throughout the summer

General Protocols

LB Preparation

  1. Add 25g Luria Broth to 1L demineralised water
  2. Autoclave

LB Agar Preparation

  1. Add 25g Luria Broth and 15g Agar to 1L demineralised water
  2. Autoclave

Rubidium Chloride Competent Cells

  1. Inoculate 1ml of cell culture (grown overnight) into flask containing Psi Broth
  2. Incubate for 2h at 37°C
  3. Transfer culture into sterile falcon tube, place in ice for 15min
  4. Centrifuge at 4000rpm for 5 minutes
  5. Discard supernatant, then add TfBI buffer and resuspend pellet
  6. Place in ice for 15 minutes
  7. Centrifuge at 4000rpm for 5 minutes
  8. Discard supernatant, then add TfBII buffer and resuspend pellet
  9. Produce 50ul aliquots

Heat-Shock Transformation

  1. Add insert DNA to cell aliquot
  2. Place on ice for 15 minutes
  3. Heat-shock: place in a 42°C heat-block for 45 seconds
  4. Place samples back on ice for 2 minutes
  5. Add LB
  6. Incubate at 37°C for 30-60 minutes
  7. Centrifuge at 4000rpm for 5 minutes
  8. Discard 300uL LB
  9. Resuspend cells in remaining 200uL LB
  10. Plate out
  11. Incubate at 37°C overnight.

80% Glycerol Preparation

  1. Add 80ml 99.7% glycerol to 20ml demineralized water
  2. Autoclave

Glycerol Stock Preparation

  1. Cultures plated on LB Agar + antibiotic and grown at 37°C overnight.
  2. A 5ml LB culture in LB+antibiotic inoculated from a single, freshly growing colony.
  3. Cultivate for 16h at 37°C, with constant shaking
  4. 0.5ml of this culture inoculated into sterile vial
  5. Add 0.5ml of 80% glycerol
  6. Vortex
  7. Spin down
  8. Freeze them at -80 degrees

QIAprep Spin Miniprep Kit

  • Materials per sample
    • 250ul P1 buffer (suspension buffer)
    • 250ul P2 buffer (Lysis buffer)
    • 350ul N3 buffer
    • 750ul PE buffer
    • 500ul PE buffer
    • Columns
  1. Spin cells down at 4000rpm for 10 minutes
  2. Discard supernatant (LB)
  3. Resuspend pellet in P1 buffer
  4. Transfer to labeled Eppendorf tube
  5. Add P2 buffer. Solution should turn blue
  6. Invert tubes 4-6 times, then wait for 2 minutes
  7. Stop the reaction by adding N3 buffer and immediately inverting 4-6 times. Solution should turn clear
  8. Centrifuge at 13000 rpm for 10 minutesv
  9. Decant/pipette supernatant into mini-prep columns. Discard flow-through
  10. Wash with PE buffer (750ul)
  11. Centrifuge at 13000 rpm for 1 minutev
  12. Discard flow-through. Add second wash of PE buffer (500ul)v
  13. Centrifuge at 13000 rpm for 1 minute
  14. Discard flow-through
  15. Centrifuge empty columns at 13000 rpm for 1 minute to eliminate any excess wash buffer
  16. Discard flow-through
  17. Move columns into a labelled eppendorf
  18. Add 30-40ul distilled water and wait for 2-3minutes
  19. Elute DNA by centrifuging at 13000 rpm for 1 minute, do not discard flow-through. Discard column.
  20. Nanodrop

Digestion

  • Materials
    • 2ul Cutsmart buffer (New England Biolabs)
    • 1ul Restriction enzymes
    • 12-16ul DNA to be digested
    • Distilled water (up to 20ul total volume)
  1. Prepare mix
  2. Incubate for 1-2h at 37 degrees

1% Agarose Gel

  • Materials
    • 1g Agarose
    • 100mL 1X TAE buffer
    • 8uL SYBR Safe
  1. Mix Agarose and 1x TAE buffer
  2. Heat up until Agarose is dissolved
  3. Add SYBR Safe
  4. Pour into gel tray and let cool

Agarose Gel Electrophoresis

  • Materials
    • 1% Agarose gel DNA ladder
    • 6x loading dye
    • Electrophoresis cuvette
  1. Set gel tray into cuvette, filled with 1x TAE buffer
  2. Inoculate samples, previously dyed with 6x loading dye. Additionally, provided a DNA ladder for further reference of DNA sizes
  3. Run gel at 110V for 30-40min

Ovenight Cell Incubation

  • Materials
    • 5mL Luria Broth
    • 5ul specific antibiotic
    • Loops (for colony picking or glycerol stock scraping)
  1. Add Luria Broth into 50mL tube
  2. Inoculate specific antibiotic
  3. Scrape/pick glycerol stock surface/colony and transfer into falcon tube
  4. Incubate at 37°C overnight

1x TAE buffer

  • 1x solution contas 40nM Tris, 20mM acetic acid, 1mM EDTA