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Team:Imperial/Protocols
From 2014.igem.org
Protocols
Here you can find the protocols we used throughout the summer
General Protocols
LB Preparation
- Add 25g Luria Broth to 1L demineralised water
- Autoclave
LB Agar Preparation
- Add 25g Luria Broth and 15g Agar to 1L demineralised water
- Autoclave
Rubidium Chloride Competent Cells
- Inoculate 1ml of cell culture (grown overnight) into flask containing Psi Broth
- Incubate for 2h at 37°C
- Transfer culture into sterile falcon tube, place in ice for 15min
- Centrifuge at 4000rpm for 5 minutes
- Discard supernatant, then add TfBI buffer and resuspend pellet
- Place in ice for 15 minutes
- Centrifuge at 4000rpm for 5 minutes
- Discard supernatant, then add TfBII buffer and resuspend pellet
- Produce 50ul aliquots
Heat-Shock Transformation
- Add insert DNA to cell aliquot
- Place on ice for 15 minutes
- Heat-shock: place in a 42°C heat-block for 45 seconds
- Place samples back on ice for 2 minutes
- Add LB
- Incubate at 37°C for 30-60 minutes
- Centrifuge at 4000rpm for 5 minutes
- Discard 300uL LB
- Resuspend cells in remaining 200uL LB
- Plate out
- Incubate at 37°C overnight.
80% Glycerol Preparation
- Add 80ml 99.7% glycerol to 20ml demineralized water
- Autoclave
Glycerol Stock Preparation
- Cultures plated on LB Agar + antibiotic and grown at 37°C overnight.
- A 5ml LB culture in LB+antibiotic inoculated from a single, freshly growing colony.
- Cultivate for 16h at 37°C, with constant shaking
- 0.5ml of this culture inoculated into sterile vial
- Add 0.5ml of 80% glycerol
- Vortex
- Spin down
- 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
- Spin cells down at 4000rpm for 10 minutes
- Discard supernatant (LB)
- Resuspend pellet in P1 buffer
- Transfer to labeled Eppendorf tube
- Add P2 buffer. Solution should turn blue
- Invert tubes 4-6 times, then wait for 2 minutes
- Stop the reaction by adding N3 buffer and immediately inverting 4-6 times. Solution should turn clear
- Centrifuge at 13000 rpm for 10 minutesv
- Decant/pipette supernatant into mini-prep columns. Discard flow-through
- Wash with PE buffer (750ul)
- Centrifuge at 13000 rpm for 1 minutev
- Discard flow-through. Add second wash of PE buffer (500ul)v
- Centrifuge at 13000 rpm for 1 minute
- Discard flow-through
- Centrifuge empty columns at 13000 rpm for 1 minute to eliminate any excess wash buffer
- Discard flow-through
- Move columns into a labelled eppendorf
- Add 30-40ul distilled water and wait for 2-3minutes
- Elute DNA by centrifuging at 13000 rpm for 1 minute, do not discard flow-through. Discard column.
- 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)
- Prepare mix
- Incubate for 1-2h at 37 degrees
1% Agarose Gel
- Materials
- 1g Agarose
- 100mL 1X TAE buffer
- 8uL SYBR Safe
- Mix Agarose and 1x TAE buffer
- Heat up until Agarose is dissolved
- Add SYBR Safe
- Pour into gel tray and let cool
Agarose Gel Electrophoresis
- Materials
- 1% Agarose gel DNA ladder
- 6x loading dye
- Electrophoresis cuvette
- Set gel tray into cuvette, filled with 1x TAE buffer
- Inoculate samples, previously dyed with 6x loading dye. Additionally, provided a DNA ladder for further reference of DNA sizes
- 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)
- Add Luria Broth into 50mL tube
- Inoculate specific antibiotic
- Scrape/pick glycerol stock surface/colony and transfer into falcon tube
- Incubate at 37°C overnight
1x TAE buffer
- 1x solution contas 40nM Tris, 20mM acetic acid, 1mM EDTA
