IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Implementation

Biofilm Detection: Implementation

• Introduction
• Specifications
• Design
• Modelling
• Implementation
• Testing/Validation
• Notes
• References

Protocols

Plasmid Preparation

Summary: multiplying plasmid by putting them into the bacteria

Making LB Medium

To make 1 L of LB medium:

• 1 L distilled water
• 10 g tryptone
• 5 g yeast extract
• 5 g NaCl
• 1 ml 1 M NaOH

1. Autoclave for 25 minutes
2. Wait until flask is hand-hot, you can hold the flask in your hand comfortably (~50°C)

[Note: Please minimize contamination to the medium during preparation and after use. If LB us suspected to be contaminated, make a new one.]

Maxiprep

1. Add up to 100 µg/ml of ampicillin and/or 50 µg/ml of tetracyclin to LB
2. Pick one colony of E. coli and use it to inoculate 5 ml of LB medium
3. Grow overnight at 37°C with vigorous shaking
4. Inoculate 500ml of LB medium with overnight culture and grow until saturation
5. Pellet cells by centrifuging at 6000 rpm at 4°C
   [Note: Pellets can be stored at -20°C if not required.]
6. Resuspend pellet in 4 mL GTE solution in a centrifuge tube
7. Add 1 ml of 25 mg/ml hen egg white lysozyme and allow it to stand 10 min at room temperature
   [Note: Plasmids are fragile, be gentle while handling and mixing solutions with plasmids]
8. Add 10 ml 0.2 M NaOH/1% SDS and mix gently until solution becomes homogeneous and clears, leave on ice for 10 min
9. Add 7.5 ml of 3 M potassium acetate solution and mix gently until a large precipitate forms, leave on ice for 10 min
10. Centrifuge at 13,000 rpm for 10 minutes at 4°C
11. Transfer supernatant into a centrifuge tube through several layers of cheesecloth to remove any floating material
    Repeat if there are still traces amount of solid material in your supernatant
12. Add 0.6 ml of isopropanol for every ml of supernatant and mix by inversion
13. Leave for 5-10 minutes at room temperature
14. Centrifuge at 11,500 rpm for 10 minutes at room temperature
15. Remove supernatant and wash pellet with 2 ml of 70% ethanol
16. Centrifuge at 11,500 rpm for several minutes
17. Remove ethanol and dry pellet

Miniprep

1. Add up to 100 µg/ml of ampicillin and/or 50 µg/ml of tetracyclin to LB
2. Pick one colony of E. coli and use it to inoculate 5 ml of LB medium
3. Grow overnight at 37°C with vigorous shaking
4. Pellet cells by microcentrifuging 1.5ml of cells at maximum speed for 20 seconds and remove supernatant
5. Resuspend pellet in 100 µl GTE solution leave for 5 min at room temperature
6. Add 200 µl 0.2 M NaOH/1% SDS solution, mix by tapping, leave on ice for 5 min.
7. Add 150 µl potassium acetate solution and vortex for 2 sec, leave ice for 5 min.
   [Note: Make sure solutions are properly mixed]
8. Centrifuge at maximum speed for 3 minutes
9. Transfer supernatant to a fresh tube and add 0.8 ml of 95% ethanol, leave for 2 minutes at room temperature
10. Centrifuge at maximum speed for 1 min
11. Remove supernatant and wash pellet with 1 ml of 70% ethanol
12. Resuspend the pellet in 30 µl TE buffer

[Note: DNA can be digested in 1 µl of a 10 mg/ml RNase if RNA contamination is suspected]

Electroporation

process of shocking the cells to make them take in the plasmid: plasmid naturally not diffusable

Plasmid Extraction

extraction of plasmid from the cell: breaking the cell and precipitate the plasmid

Restriction Digest

enzymes that cut on specific sites on the DNA

Ligation

sticking the DNA together

Agarose Electrophoresis

method to seperate DNA of different sizes, using an electric field, in a gel

Materials

• Electrophoresis buffer
• DNA dye
• Electrophoresis-grade agarose
• 10× loading buffer
• DNA molecular weight markers
• 55°C water bath

Apparatus

• Gel casting platform
• Gel combs (slot formers)
• DC power supply
• Horizontal gel electrophoresis apparatus

Protocols

Preparing the gel

• Prepare an adequate volume of electrophoresis buffer to fill the electrophoresis tank
• Add electrophoresis-grade agarose to a volume of electrophoresis buffer ~ 1.2% of the gel
• Melt the agarose a microwave oven or autoclave and swirl to ensure even mixing
  • NOTE: melted agarose must be cooled to 55°C in a water bath before pouring onto the gel platform
• Add the reagent to visualise DNA
• Seal the gel casting platform if it is open at the ends. Pour in the melted agarose and insert the gel comb, making sure that no bubbles are trapped underneath the combs and all bubbles on the surface of the agarose are removed before the gel sets

Loading and running the gel

• After the gel has hardened, remove the tape from the open ends of the gel platform and withdraw the gel comb
• Place the gel casting platform containing the set gel in the electrophoresis tank
• Add sufficient electrophoresis buffer to cover the gel, until the tops of the wells are submerged. Make sure no air pockets are trapped within the wells
• Load the DNA into the wells with a pipettor or micropipet
  • NOTE: include appropriate DNA molecular weight markers
• Set the voltage to the desired level, typically 1 to 10 V/cm of gel, to begin electrophoresis. The progress of the separation can be monitored by the migration of the dyes in the loading buffer
• Turn off the power supply when the bromphenol blue dye from the loading buffer has migrated a distance judged sufficient for separation of the DNA fragments

DNA Extraction/Purification

extract the DNA from the gel

Biobricks

• Construct, clone and growth over 2 day period