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From 2011.igem.org

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Protocols

This page lists all the protocols used in our project. We have classified them into five main categories as follow.

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General

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Antibiotics

Four different antibiotics (kanamycin, chloramphenicol, ampicillin & tetracycline) have been used during the course of the project. They have been used in following working concentrations, unless stated otherwise:

- Kanamycin - 35 µg/ml

- Chloramphenicol – 35 µg/ml

- Tetracycline – 35 µg/ml

- Ampicillin – 100 µg/ml

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Cell transformation

- Remove competent cells from freezer and allow to thaw on ice for 10 min

- Add 3-5 μl of DNA to the cells

- Incubate on ice for 20 min

- Heat shock the cells at 42°C for 45 seconds

- Add 500 μl of LB Broth, and incubate in a shaker at 37°C for 1 hour

- Centrifuge for 10 min, remove and keep 100 μl of the supernatant, and pour away the rest.

- Re-suspend the pellet in the 100 μl of supernatant that was removed earlier

- Spread 5 μl onto one agar plate, and then pour the rest onto another. Incubate the plates overnight at 37°C.

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DNA rehydration

- Centrifuge DNA for 4 minutes to ensure DNA is at the bottom of the tube

- Consult the Synthesis Report to work out how much water to add - multiply the yield in nmol by 10, and that value is the volume of water in µl

- Gently pipette up and down, then leave for 5 minutes

- Vortex, pipette up and down, and then leave for a further 5 minutes

- Take 10µl of this DNA solution and add it to 90µl of pure water to make a working solution

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Colony PCR

- 20.75 μl H2O

- 2.50 μl Pfu Buffer (10x)

- 0.25 μl dNTP mix (40 mM)

- 0.50 μl primer 1 (100 ng/ul)

- 0.50 μl primer 2 (100 ng/ul)

- 0.50 μl Pfu Cx polymerase (2.5 U/ul)

Make up a master mix of everything except polymerase and aliquot into PCR tubes. Pick colonies from your plate, spot onto your reference grid plate and then pipette up and down in the PCR tube. The high denaturation temperature of PCR lyses the cells so that the DNA contents can be used as template for sequencing primers to anneal to and polymerase to extend, so you can verify that the colonies contain your plasmid based on analytical gel electrophoresis.

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CPEC assembly

Make up 50 μl reactions with 5 μl DNA total with each part to be assembled at equimolar concentrations (i.e. make appropriate dilutions)

- 33.5 μl ddH2O

- 10 μl Phusion HF buffer (5x)

- 1 μl dNTP mix (40 mM)

- 5 μl DNA (1-50 ng)

- 0.5 Phusion high fidelity DNA polymerase (2 U/μl^-1)

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Extracting fluorescent protein samples

- Colonies were picked from the plates containing the transformants, and these were grown in 5ml of LB Broth for 6 hours at 37°C. These starter cultures were then used to inoculate 100 ml flasks of broth which were grown overnight on a shaker at 37°C.

- The cultures were transferred into 50ml Falcon tubes, and spun down in a centrifuge for 10 min at 5000 rpm. The supernatant was poured off, and the pellet re-suspended in 20mM Tris Lysis buffer.

- The cell suspension was sonicated to lyse the cells, and then the samples were centrifuged again to collect cell debris. The supernatant was collected and stored in a refrigerator at 4°C. Do not freeze the samples, as this may cause protein degradation

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Thermostability assay

- Take 50ul of fluorescent protein sample and heat for 2 hours at the following temperatures (degrees centigrade) in a PCR thermocycler. 35.3; 37.1; 39.7; 44.1; 48.5; 52.5; 56.7; 57.1; 59.1; 61.3; 61.7; 65.7; 66.3; 69.7; 70.9; 72.5; 74.1; 75.3; 79.1; 83.7; 88.8; 92.3; 95.1; 96.5

- Dilute 30ul of heat treated samples in 170ul of lysis buffer and measure fluorescence on plate reader.

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SOB/SOC

To make 1 litre of Super Optimal Broth (SOB):

- 20 g peptone

- 5 g yeast extract

- 0.58 g NaCl

- 0.44 g KCl

- 0.95 g MgCl₂

- 1.2 g MgSO₄

- Add the above ingredients to a flask, and then make up to 1 litre with distilled water

To make a litre of SOC, first make up a litre of SOB and then add:

- 3.6 g Glucose

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Glycerol stock preparation

- Take 0.8ml of cell suspension
- Add 0.2 of 80% glycerol in each eppendorf.
- Mix bacteria in 80% glycerol by pipetting up and down.
- Freeze at -80°C.

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