Growth Media

Lysogeny Broth

Materials

• 10g of tryptone
• 5g of yeast extract
• 10g of NaCl
• 1L of Deionized Water
• 1M NaCl
• 1M KOH

Procedure

1. Use a container with at least double the volume of the liquid that you are making.
2. Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 mL deionized water.
3. Adjust the pH of the medium to 7.0 using 1M NaOH or KOH and bring volume up to 1 liter.
4. Autoclave.
5. Store at room temperature or +4°C.

X-Gal/IPTG Protocol

1. Add the following to 200 mL of melted (~55°C) LB-Agar
• 20 uL X-Gal (20mg/ml)
• 20 uL IPTG (100mM)
• 20 uL of appropriate antibiotic
2. Swirl to mix, try to avoid making bubbles
3. Dry at room temp and wrap in foil to prevent degradation

Gel Electrophoresis

10X TAE (Tris Acetate EDTA) Buffer

1. Dissolve the following in 600mL of dH₂O
• 48.4 g Tris base (FW 121)
• 11.42 mL glacial acetic acid
• 40 mL of 0.25M EDTA (pH 8.0)
2. Make up volume to 1.0 L with dH₂O
3. Autoclave in appropriately sized bottle

Competent Cells

Materials

• LB media (in 50 mL in 250 conical flask) pre- autoclaved (x2)
• Overnight culture of DH5α (x2)
• Shaker in 37°C room
• 15 mL falcon tubes (~x15)
• Eppendorf tubes (1 mL)
• 50% glycerol
• 0.1 M CaCl2
• Waste bucket with Vircon (kill bacteria)
• Ice buckets

Protocol

1. Inoculate 1% inoculum from overnight culture
2. 1 mL for 100 mL medium
3. 2 separate 250 mL flasks each with 50 mL LB
4. 0.5 mL of culture- grow with shaking until ~ 0.375 OD (at 600 nm) - we went a bit over, but other protocols say this is ok
5. Put flasks on ice for 10 minutes
6. Put 10 mL of inoculum into 15 mL falcon tubes (repeat to use up all the inoculum)
7. Centrifuge @ 5K for 10 minutes @ 4°C
8. Discard supernatant and resuspend pellet in 2 mL of 0.1 M CaCl2
9. Chill on ice for 20 mins
10. Centrifuge @ 5K for 10 minutes @ 4°C
11. Discard supernatant and resuspend each pellet in 0.28 mL of 0.1 M CaCl2 and 0.12 mL of 50% glycerol - you can also prepare a solution of CaCl2 and glycerol before hand
12. Aliquot 100 μL of resuspension into sterile eppendorfs
13. Store at -80°C

PCR

Colony PCR Protocols

For PCR reactions up to 50 μL add the following to each PCR Tube:

Substance	GoTaq	Q5	Phusion
Forward Primer	2.5	2.5	2.5
Reverse Primer	2.5	2.5	2.5
Polmerase	.2	25 μL mastermix	.5
dNTPs	.5	-	1
dH2O	14.3	18.0	33.5
Buffer	5	-	10
DNA	touch of colony	touch of colony	touch of colony

PCR Machine Cycling Times and Temperatures: E. coli and Sinorhizobium Plasmids

Load on 1% agarose gels and run electrophoresis tank until the coloured bands are near the bottom of the gel. Use U:genius machine to photograph and visualise bands.

Digestion Protocols

For each of the following digestions add:

1. 1 uL Restriction Enzyme
2. 1 ug DNA
3. 5 uL 10X NEBuffer
4. dH₂O to make up 50 uL volume

• For the digestions with SpeI, XbaI, PstI and EcoRI-HF, incubation time is 5-15 minutes at 37°C.
• Inactivation of XbaI and EcoRI-HF takes 20 minuties at 65°C.
• SpeI and PstI are inactivated at 80°C for 20 minutes.
• SpeI, XbaI ,and EcoRI-HF have 100% Buffer activity with either NEBuffer 2.1 or Cutsmart while PstI digestions work best with 3.1

Clarification of lysate

1. Centrifuge for 5 minutes at 11,000 x g at room temperature
2. Put 500μl of Buffer PW1 per 1.5ml microcentrifuge tube used in heat block heated to 50^օC

Bind DNA

1. Place ISOLATE II Plasmid Mini Spin Column in a 2ml Collection Tube
2. Pipette a maximum of 750μl of clarified sample supernatant onto column
3. Incubate at room temperature for 2 minutes.
4. Centrifuge for 1 minute at 11,000 x g and discard flow-through.
5. Repeat stage 4 using the same ISOLATE II Plasmid Mini Spin Column and 2ml Collection Tube with the clarified sample supernatant from the other 1.5ml microcentrifuge tube from the same sample.

Wash silica membrane

1. Add 500μl Wash Buffer Pw1
2. Centrifuge for 1 minute at 11,000 x g
3. Add 600μl Wash Buffer PW2 (supplemented with ethanol)
4. Centrifuge for 1 minute at 11,000 x g
5. Discard flow-through and reuse Collection Tube

Dry silica membrane

1. Centrifuge for 2 minutes at 11,000 x g, to remove residual ethanol
2. Place ISOLATE II Plasmid Mini Spin Column in a 1.5ml microcentrifuge tube.

Elute DNA

1. Add 50μl Elution Buffer P directly on the top of the silicon matrix
2. Incubate at room temperature for 2 minutes
3. Centrifuge for one minute at 11,000 x g.

Phosphate Assay

Reagent preparation

• Phosphate Reagent: 15 ml of colorimetric dye. Ready to use as supplied. Equilibrate to room temperature before use. There may be a small amount of precipitate visible which does not affect the assay performance. Store at room temperature. Actually orange.
• Phosphate Standard: 500uL of 10mM Phosphate standard. Ready to use as supplied. Equilibrate to room temperature before use. Store at room temperature. Actually Clear.

Standard preparation

Materials

• Phosphate Standard
• Phosphate Reagent
• Distilled Water

Procedure

1. Dilute 5ul of supplied Phosphate Standard into 495ul of dH2O in a 1.5ml Eppie. Label ‘S’
2. Make further dilutions to construct the curve. Suggested values are below. Make these in 1.5ml Eppendorfs

Volume S/ ul	Volume dH2O/ ul	Concentration/ nMol/200ul(well)
0	600	0
30	570	1
60	540	2
90	510	3
120	480	4
150	450	5

1. Pipette 200ul of each curve sample into a well.
2. Add 30ul of Phosphate Reagent to each well
3. Leave to equilibrate for 30 mins at room temperature. N.B the reagent is light sensitive so store in a dark room while equilbrating

Sample preparation

N.B. Make sure our sample is diluted to ensure the readings are within the standard value range.

Steps for cell (adherent or suspension) samples:

1. Harvest the amount of cells necessary for each assay (initial recommendation = 2 x 10^6 cells).
2. Wash cells with cold TBS (kept in 4oC fridge, stable for 3 months).
3. Resuspend the cell pellet in 150 µL of Assay Buffer (TBS) on ice.
4. Homogenize cells quickly by pipetting up and down a few times.
5. Sonicate cells for 50 seconds 3X at high setup (one cycle = 30 s sonication – 10 s break – 10 s sonication – 10 s break).
6. Centrifuge sample for 15 minutes at 4°C at top speed using a cold microcentrifuge to remove any insoluble material.
7. Collect supernatant and transfer to a clean tube.

• Keep on ice

Tris-Buffered Saline (TBS) Recipe

1. 50 mM Tris-Cl, pH 7.5
2. 150 mM NaCl
3. To prepare, dissolve 6.05 g and 8.76 g NaCl in 800 mL of H2O.
4. Adjust pH to 7.5 with 1 M HCl
5. Tris Make volume up to 1 L with H2O.

N.B. TBS is stable at 4°C for 3 mo.

Assay procedure and detection

Equilibrate all materials and prepared reagents to room temperature prior to use. It is recommended to assay all standards, controls and samples in duplicate.

Set up Reaction wells:

1. Standard wells = 200 µL standard dilution.
2. Sample wells = 1 – 100 µL samples (adjust volume to 200 µL/well with ddH2O).
3. Background control sample wells = 1 – 100 samples (adjust volume to 200 µL/well with ddH2O).
4. Add 30 µL of Phosphate Reagent to standard, sample and background control wells.
5. Mix and incubate at room temperature for 30 minutes protected from light.
6. Measure output at OD = 650 nm on a microplate reader.

Glassmilk recipe

1. Stir 400g silica 325 in 800 ml of dH2O
2. Leave to settle for 90 minutes
3. Remove supernatant and centrifuge 6000 rpm for 10 minutes
4. Resuspend pellet in 250 ml dH2O
5. Add conc. Nitric Acid to 50%
6. Stir and heat to a boil gently
7. Stir to RT gently
8. Centrifuge @ 6000 rpm for 10 minutes (make sure you use either glass or polypropylene tubes, polystyrene tubes can’t withstand over 3000g)
9. Resuspend in 250 ml dH2O
10. Repeat spinning and washing until pH is neutral
11. Resuspend pellet to make 50% by volume slurry

Aliquot and store indefinitely at RT

Growth Assay - Plate Reader

Materials

• 96 well plate
• 10x MOPS Media (dilute to 1X)
• K₂HPO₄ (dipotassium phosphate)
1. @0.1 mM for low P
2. @1.32 mM for high P - suggested by MOPS media recipe
• 4 mg/mL glucose
• Strains of bacteria

Purpose

Used to measure bacterial density by tracking absorbance at intervals of 30 minutes for 48 hours (220 rpm, 37°C)

Procedure

Day 1

1. Prepare the phosophate concentrations and filter sterilize them
2. Prepare the glucose solution and filter sterilize them
3. Prepare 1X MOPS (from 10X solution) filter sterilize them
4. Prepare overnights
1. 5ml of LB in a BD falcon tube
2. Take a colony off the plate with a sterile toothpick and put in falcon tube
3. Leave overnight at 37°C in a shaker

Day 2

1. Transfer overnight culture into 1.5 mL eppendorf tubes
2. Spin down overnights - 3 min at 4K
3. Wash out with MOPS and resuspend with 1 mL MOPS
4. Repeat spin step and resuspend again
5. Measure OD₆₅₀ of a 10 fold dilution of cells with a spectrophotometer blanked with MOPS
6. Calculate the amount of inocula required for a final OD₆₅₀ of 0.4 in 500 uL
7. Set up wells - 2 uL phosphate, 5 uL cells, 193 uL MOPS