Antibiotic	Concentration(μg/ml)
Ampicillin	100
Chloramphenicol	25
Tetracycline	20

Primer Name	Nucleotide Sequence
pSB1C3 seq f	caactcgagtgccacctg
PSB1C3 seq r	gcctttttacggttcctg
TliA seq f	gcagatctgagcaccagc
TliA seq r	gtccagagcacgaaagac
KERUS seq f	ggtgatggtcaggatagc
KERUS seq r	ctgccacaacaacaacac

GGTGATGGTCAGGATAGC

CTGCCACAACAACAACAC

pSB1C3 seq f: caactcgagtgccacctg – 18-mer, 3412...3429, 56degC

pSB1C3 seq r: gcctttttacggttcctg, 18-mer, 1731...1748, 52degC

TliA seq primer f: gcagatctgagcaccagc 18-mer, 711...728, 56degC

TliA seq r: gtccagagcacgaaagac – 18-mer, 831...848, 54degC

Luria-Bertani (LB) Broth

LB Broth was made using a ready mix powder from Sigma, 20g/L in ultrapure water. This consists of: 10 g/L Tryptone; 5 g/L Yeast Extract; 5 g/L NaCl. Autoclaved at 121 oC for 15 min to sterilise.

Luria-Bertani (LB) Agar

LB agar was made using a ready mix powder from Sigma, 35 g/L ultrapure water. This consists of 15 g/L Agar and as in LB Broth. Autoclaved at 121 oC for 15 min to sterilise.

5 x M9

5 x M9 was made using the following up to 1 L of ultrapure water: 64 g Na2HPO4-4H2O; 15 g KH2PO4; 2.5 g NaCl.

Working stock, 1 X M9 was made by adding into up to 1 L of ultrapure water: 200 ml of 5 x M9; 2 ml 1M MgSO4; 20 ml 20% glucose/0.4% Glycerol; 100 μl 1M CaCl2

50 x TAE

50 x TAE was made up with: 121 g Tris Base (Trizma); 28.55 gl acetic acid; 9.3 g EDTA made up in 500 ml ultrapure water.

Working stock, 1 x TAE was made by diluting 10 ml in 500 ml ultrapure water.

10 x TBS

10 x TBS was made up with: 500 ml 1M Tris.HCl, pH 7.4; 300 mM NaCl made up to 1L with ultrapure water.

Working stock, 1 x TBS by diluting 100 ml 10 x TBS up to 1000 ml with ultrapure water.

1 x PBS

1 x PBS was made up using Phosphate buffered saline tablets from Sigma.

One tablet dissolved in 200 mL of deionized water yields 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4, at 25 °C.

Upper Tris Buffer

Upper Tris Buffer was made up by adding 6.05 g Tris Base in 100 ml deionised water, pH 6.8. Add 0.4 g SDS.

Lower Tris Buffer

Lower Tris Buffer was made up by adding 36.4 g Tris in 100 ml deionised water, pH8.8. Add 0.8 g SDS.

10 x SDS Running Buffer

10 x SDS running buffer was made up by adding: 30.3 g Tris Base; 144.1 g Glycine; 10 g SDS to 1 L deionised water to pH 8.3.

Working concentration, 1 x SDS Running Buffer is made by adding 100 ml of 10 x SDS Running buffer to 10 ml deionised water.

Solubilising lipids

A 10% stock solution of triton-x100 was made by adding 1ml of Triton to 9ml sterile (autoclaved) water and incubated at 37oC, 150 rpm until solubilised.

Filter sterilise and add 10% Triton to Olive oil/Oleic acid in a 2:1 ration. Incubate at 37oC, 150 rpm to solubilise.

Materials and Equipment

Stripette, bunsen burner, sterile loop, falcon tubes, LB broth.

Time

Prep: 10 minutes
Run: 16 hours

Procedure

1. Use a stripette to take 5ml of LB broth from a 250ml conical flask.
2. Dispense into a falcon tube.
3. Sterilise a metal loop in a flame.
4. Take a culture from an agar plate using the sterile loop and put into one of the falcon tubes.
5. Agitate.
6. Replicate this using scrapings from a clean agar plate and a fresh tube to use as a positive control.
7. Put the tubes into the incubator overnight (37c, 150rpm) to grow up the cultures.

Materials and Equipment

LB broth, starter culture, P1000, P200, pipette tips, incubator, cuvettes, spectrophotometer, Virkon, falcon tubes, ice, weighing scales, centrifuge, CaCl2, 20% glycerol, stripette, eppendorf tubes and -80°C freezer.

Time

Prep: 40 minutes
Run: 5 hours

Procedure

1. Grow cells
   1. Take 1ml starter culture and add to 100ml LB broth.
   2. Incubate at 37°C, 150rpm.
   3. Check every hour by testing the optical density at 600nm (OD) using a spectrophotometer to determine whether enough cells are present in the culture.
   4. 0.600 OD is ideal, this is the point at which the cells are in the exponential growth phase.
   5. Take 1ml of culture into a cuvette to measure; dispose of this after use in Virkon.
2. Remove cells
   1. Pour 30ml aliquots from the flasks into falcon tubes; 3 tubes per flask.
   2. Put tubes on ice for approx 10mins; all equipment used from this point on must be cold e.g. pipette tips.
   3. Weigh the falcon tubes and pair together similar weight tubes for balance in the centrifuge; tubes paired together must weigh within 0.5g of each other.
   4. Spin at 4°C, 4000rpm for 5mins.
   5. After the tubes have all been spun, pour off the supernatant to remove the LB broth, leaving cells in a pellet. Put tubes back on the ice.
3. Make cells competent
   1. Add 1ml of CaCl2 to the cells, use the pipette to pull the liquid and cells up and down to resuspend.
   2. Once resuspended, add another 14ml of CaCl2; 15ml total volume.
   3. Put back on ice for approx. 10mins to allow cells to acclimatise at the temperature with the CaCl2.
   4. Re-weigh and pairs tubes again for balance.
   5. Spin again at 4°C, 4000rpm for 5mins.
   6. After tubes have been spun, leave on ice for 5mins. Pour off the supernatant?
4. Aliquot
   1. Add 600μl of 20% glycerol to each falcon tube.
   2. Label eppendorf tubes.
   3. Aliquot 200μl from each falcon tube into eppendorf tubes (3 per falcon).
   4. Freeze at -80°C.

Materials and Equipment

Ice, starter cultures, mini-prep kit, centrifuge, P100 pipette, weighing scale, Virkon.

Time

Prep: ?
Run: ?

Procedure

1. Extract cells
   1. Match up weights of falcon tubes so they are paired within 0.5g.
   2. Spin down starter cultures in a centrifuge; 5mins, 4°C, 4000rpm.
   3. Pour off supernatant into Virkon.
2. Resuspend cells
   1. Add 250μl of P1 resuspension buffer to each falcon tube using P1000.
   2. Resuspend the cells.
   3. Use pipette to move suspension into separate, labelled eppendorf tubes.
3. Lyse cells
   1. Add 250μl of P2 buffer to each eppendorf tube.
   2. This will lyse cells - a blue colour will indicate they have been lysed.
   3. Do not leave for more than 5mins.
4. Neutralise cells
   1. Add 350μl of N3 neutralisation buffer.
   2. Once the reaction is complete, the liquid will turn clear/white.
5. Purify DNA
   1. Spin down the cells in a centrifuge; 10 mins, 17000g, 4°C.
   2. Pour supernatant into mini-prep columns.
   3. Centrifuge columns for 1 min, 17000g, 4°C.
   4. Discard flow through.
   5. Add 500μl of PB buffer to each column.
   6. Centrifuge columns for 1min, 17000g, 4°C.
   7. Discard flow through.
   8. Add 750μl PE buffer to each column.
   9. Centrifuge columns for 1min, 17000g, 4°C.
   10. Pour off supernatent.
   11. Centrifuge columns for 1min, 17000g, 4°C.
   12. Discard bottom of the mini-prep.
   13. Move column to a new labelled eppendorf.
   14. Add 50μl of elution buffer.
   15. Centrifuge columns for 1min, 17000g, 4°C.
   16. Discard column.
   17. Immediately place on ice; store in the B56 sewer sample freeze.

Materials and Equipment

Agarose mix, TAE buffer, Flask, Microwave, Ethidium bromide, P10, Tips, Autoclave tape, Gel tray, Comb, Buffer tray, Loading buffer, Dna, Eppendorf, Transilluminator.

Time

Prep: 30 mintues
Run: 1 hour

Procedure

1. Make up a 1% agarose gel
   1. Weigh out 0.4g agarose.
   2. Measure 40ml TAE buffer.
   3. Add both into a 250ml conical flask.
   4. Swirl.
   5. Microwave on full for 2 minute, swirling at intervals to ensure all the agarose has dissolved.
   6. Run side of flask under tap to cool, until comfortable to hold in a gloved hand.
2. Prepare the gel
   1. Add 1μl ethidium bromide to the conical flask containing the melted agarose.
   2. Pour this solution smoothly into a gel tray (sealed with autoclave tape) and add a comb which allows for either 8 or 13 DNA samples to be run at once.
   3. Leave the gel to set for approximately 15 - 30 minutes.
   4. Once the gel is set remove the comb and autoclave tape.
   5. Place the gel tray inside the buffer tray and fill the remaining space with TAE buffer.
3. Load 'checking' samples
   1. Load 5μl of 1kn ladder into the first well of the gel.
   2. Pipette 2μl 5x loading buffer onto a sheet of parafilm.
   3. pipette 8μl DNA onto the loading buffer and pipette up and down to mix.
   4. Resting the pipette tip on the back of the well, load 8μl of sample.
   5. Repeat for each sample.
   6. Run the Gel at 100v for 60 minutes.
4. Load 'extraction' samples
   1. Load 5μl of 1kn ladder into the first well of the gel.
   2. Add 5x loading buffer into each sample, to give a final ratio of 1:4.
   3. Resting the pipette tip on the back of the well, load as much sample as possible.
   4. Repeat for each sample.
5. Visualise
   1. Remove the agarose gel from the gel tray and place in the trans-illuminator.
   2. Open UVP (on the computer desktop).
   3. Select the type of gel and the exposure time.
   4. Press capture to take an image.
   5. Save image to the iGEM2014 folder.

Materials and Equipment

Sterile hood, Flask, Agar mix, dH2O, antibiotic, empty plates, P200, tips.

Time

Prep: 15 minutes
Run: 20 minutes

Procedure

1. Use the sterile hood.
2. Make up 100ml LB Agar (Protocol 2).
3. Heat in the microwave as follows:
   • Power setting 4 for 3 minutes.
   • Wait for 6 minutes.
   • Power setting 4 for 2 minutes.
4. Wait to cool (until the flask is warm but comfortable to hold).
5. Add 100μl 1/1000 stock of appropriate antibiotic to the agar.
6. Pour 4 plates and leave inside the hood to cool and dry.

Materials and Equipment

Nanodrop, P10, Tips, dH2O, Paper towel.

Time

Prep: 5 minutes
Run: 5 minutes

Procedure

1. Set up the NanoDrop2000, select “Nucleic Acid”, then “Routine Verification”.
2. Spray paper towel with distilled water, then clean metal nodules on metal part and lid.
3. Add 1μl of buffer solution to nodule.
4. Close lid.
5. Select 'Blank' on the programme.
6. Reopen lid.
7. Add 2μl of DNA Sample.
8. Close lid.
9. Select 'measure' on the programme.
10. Take note of the concentration (in ng/ml).
11. Clean nodules again with distilled water.
12. Repeat for different DNA sample.