Team:Wash U/Japanese/Protocol

Procedures
Contents
 * 1) Polymerase Chain Reaction (PCR)
 * 2) Transformation
 * 3) Mini-Prep
 * 4) BioBrick Assembly
 * 5) Digestion
 * 6) Ligation
 * 7) Gel Electrophoresis
 * 8) PCB Extraction
 * 9) Glycerol Stock Solution

Polymerase Chain Reaction (PCR)
Great resource for designing primers for biobrick parts: Primer Design
 * general description

Materials
 * list of materials

Procedure
 * list of steps

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Transformation

 * General description

Materials
 * List of materials

Procedures
 * 1) Start thawing the competent cells on crushed ice.
 * 2) Add 50 µL of thawed competent cells and then 1 - 2 µL of the resuspended DNA to the labelled tubes. Make sure to   keep the competent cells on ice.
 * 3) Incubate the cells on ice for 30 minutes.
 * 4) Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath improves heat transfer to the cells.
 * 5) Incubate the cells on ice for 5 minutes.
 * 6) Add 200 μl of SOC broth (make sure that the broth does not contain antibiotics and is not contaminated)
 * 7) Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.
 * 8) Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
 * 9) Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin because the resistance enzyme is excreted by the bacteria, and inactivate the antibiotic outside of the bacteria.
 * Note: Restriction sites E=EcoR1-HF; X=Xba1; S=Spe1; P=Pst1; M=Mixed Site
 * To view the full BioBrick Manual procedures, please click here.

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Mini-Prep

 * general description

Materials
 * List of materials

Procedures
 * List of Procedures

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BioBrick Assembly

 * General description

Materials
 * List of materials

Procedures
 * 1) Begin BioBrick Assembly with three separate plasmids, an upstream part, a down stream part, and a destination plasmid.  It is important that the destination plasmid contain the toxic gene ccdB in the BioBrick cloning site and a different antibiotic resistance to the upstream and downstream parts.
 * 2) Digest each part with the proper restriction enzymes to isolate the upstream and downstream parts, and to remove the BioBrick from the destination plasmid.
 * 3) Ligate the products.  The result will be the upstream part connected to the downstream part in the destination plasmid.  This new composite part can then be used to transform competent cells.
 * To view the full Biobrick Assembly manual, please click here.

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Digestion

 * General Description

Materials
 * list of materials

Procedures
 * Note: The procedures from this point forward assume that DNA has been amplified and purified. If this is not the case, please read the Polymerase Chain Reaction procedure for amplification and the Mini-Prep procedure for purification.

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 * 1) Begin by thawing the upstream, downstream, and destination plasmid parts along with the NEBuffer 2 and BSA.
 * 2) In three separate PCR microcentrifuge tubes labeled upstream, downstream, and destination, add 500ng of the respective dried DNA and dilute with dH20 to 42.5 uL.
 * 3) Add 5 uL of NEBuffer 2 and 0.5 uL of BSA to each tube.
 * 4) Add 1 uL of the first appropriate enzyme to each tube.  Then add 1 uL of the second appropriate enzyme.
 * 5) Flick each tube to mix reagents and incubate at 37C for 15 minutes.
 * 6) Transfer the tubes to an incubator set at 80C for another 20 minutes.  This step will deactivate the restriction enzymes.
 * 7) Digestion is now finished and products should be stored at -20C.

Ligation

 * General description

Materials
 * List of Materials

Procedures
 * Note: this procedure requires the products of a successful digestion.

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 * 1) Thaw the 10X T4 DNA Ligase Reaction Buffer and mix to dissolve the precipitate.
 * 2) Add 11 uL of dH2O to a 200 uL PCR tube.  Then add 2 uL of each of the digestion products (upstream, downstream and destination) together into this new tube.
 * 3) Add 2 uL of 10X T4 DNA Ligase Reaction Buffer to the 200 uL PCR tube.
 * 4) Add 1 uL DNA Ligase to the PCR tube and flick to ensure the contents are mized.
 * 5) Let the mix stand for 10 minutes at room temperature before incubating at 80C for 20 minutes (deactivates enzymes).
 * 6) Store the products at -20C until they are needed for a transformation.

Gel Electrophoresis

 * General Description

Materials
 * List of Materials

Procedures
 * text

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PCB Extraction

 * General Description

Materials
 * Spirulina Powder
 * dI water
 * Methanol
 * HgCl2
 * Trifluoroacetic acid (TFA)
 * Trichloroacetic acid (TCA)
 * Acetonitrile

Procedures
 * 1) Rehydrate Spirulina powder in dI water (30ml/g dry weight) for 10 min.
 * 2) Centrifuge at 30,000xg for 20 min, decant and save supernatant.
 * 3) Precipitate phycocyanin from supernatant with 1% (w/v) TCA by incubation for 1h at 4oC in the dark.
 * 4) Collect by centrifugation at 30,000xg for 20 min.
 * 5) Wash with methanol (2x 20ml/g Spirulina powder).
 * 6) Resuspend blue pellet in methanol (2ml/g powder) containing 1mg/ml HgCl2.
 * 7) Incubate for 20h at 42oC in the dark.
 * 8) Remove protein by centrifugation at 10,000xg for 10 min.
 * 9) Precipitate mercuric ion with 2-Mercaptoethanol (1ul/ml), remove by centrifugation at 30,000xg for 10 min.
 * 10) Dilute bilin mixture 10-fold with 0.1% (v/v) TFA.
 * 11) Apply to C18 Sep-Pak cartridge.
 * 12) Wash cartridge with 0.1% (v/v)TFA (2x 3ml) and acetonitrile/0.1% TFA (20:80; 2x 2ml.
 * 13) Elute bilin with 3ml acetonitriel/0.1% TFA (60:40) and dry in vacuo.
 * Note:Expected yields are 4 umol PCB per 6g Spirulina
 * Thanks to Dr. Clark Lagarias for providing this protocol. ref

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Glycerol Stock Solution
Materials


 * 1) Centrifuge/screw top tubes
 * 2) 50% glycerol sol
 * 3) LB Cultures

Procedures
 * 1) Label centrifuge/screw top tubes
 * 2) Pipet 300 µl of 50% glycerol solution into each tube
 * 3) Pipet 700 µl of LB culture into each tube
 * 4) Close & vortex to mix.
 * 5) Put in -80 C.

Recipes
Lysogeny Broth (LB) Media ATCC medium: Van Niel's yeast agar Super Optimal Broth (SOB) Super Optimal Broth with Catabolite repression (SOC) 50X TAE Buffer 5X TBE Buffer Tris EDTA (TE) buffer 5X 1L Agarose Gel (for electrophoresis of DNA >100bp) Back To Top
 * 10g Tryptone
 * 5g Yeast Extract
 * 10g NaCl
 * 15g Agar
 * up to 1 Liter H20
 * adjust pH to 7.5
 * 1.0 g K2HPO4
 * 0.5 g MgSO4
 * 10.0 g yeast extract
 * 20.0 g agar
 * 1.0 L tap water
 * Adjust pH to 7.0-7.2
 * 2% w/v bacto-tryptone (20g)
 * 0.5% w/v bacto-yeast extract (5g)
 * 8.56mM NaCl (0.5g) or 10mM NaCl (0.584g)
 * 2.5mM KCl (0.186g)
 * ddH2O to 1L
 * In addition to all of the SOB components,
 * 10mM MgCl2 (0.952g)
 * 20mM glucose (3.603g)
 * 242 g Tris base (2-amino-2-hydroxymethyl-propane-1,3-diol) (= 2 mole)
 * 57.1 ml glacial acetic acid (= 100% acetic acid) (57.19 ml = 1 mole)
 * 100 ml 0.5 M Na2 EDTA (pH 8.0)
 * ddH2O to 1L
 * 53 g of Tris base (CAS# 37186)
 * 27.5 g of boric acid (CAS# 11280)
 * 20 ml of 0.5 M EDTA (CAS# 60004) (pH 8.0)
 * ddH2O to 1L
 * 750 mL d-H20
 * 242 g Tris base
 * 57.1 mL glacial acetic acid
 * 100 mL 0.5M EDTA (93.05 g EDTA in 500 mL d-H20, pH ~8.0)
 * fill to 1 L
 * adjust pH to 8.5
 * 50 mL 1X TAE buffer
 * 0.8 g agarose
 * 2.5 microliters EtBr (Caution: EtBr is a known carcinogen)
 * Note: Jacob suggested adding 1.0 microliter EtBr to gel and 3.0 microliters to TAE buffer in rig.