Team:York/Protocols


Protocols

  • Click to view:
  • Growth Media
  • Gel Electrophoresis
  • Competent Cells
  • PCR
  • Digestion
  • Mini-Prep/Plasmid Purification
  • Gibson Assembly
  • Phosphate Assays
  • Growth Assays

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.

LB Agar

Procedure

  1. Follow steps to make lysogeny broth as above
  2. Add 15g of agar powder per litre of LB
  3. Autoclave in 200 or 250 mL aliquots in 500mL duran bottles
  4. Add antibiotic desired to melted agar (~55°C)
  5. Pour into petri dishes and leave to harden in asceptic fume hood
  6. Invert, label, wrap with parafilm and store at 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

Agarose Gel Electrophoresis

  1. Add 1g of agarose per 100 mL of TAE (1X) buffer
  2. Microwave to dissolve
  3. Add 10 uL of Syber-Safe per 100 mL of TAE buffer
  4. Pour into gel tray and allow to set (don't forget the comb!)
  5. Put gel into the tank and fill tank to cover top of gel
  6. Load ladder and samples and run!

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

Step Temperature Time
Initial Denaturation 95 5:00 min


30 Cycles		 Denaturation 95 15 sec
Annealing 58 15 sec
Extension 72 2-4 (1 min per kb)
Final Extension 72 5-10 min

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

Mini-Prep or Plasmid Purification

  • Harvest bacterial cells
    1. Pellet 20ml of saturated E. coli for 60 seconds at 11,000 x g.
    2. Discard supernatant and remove as much liquid as possible.
  • Lyse cells
    1. Add 500ml resuspension buffer P1 and resuspend cell pellet by vortexing.
    2. Split the solution into two 1.5ml microcentrifuge tubes.
    3. Add 250μl lysis buffer 2.
    4. Mix gently by inverting tube 8 times.
    5. Incubate at room temperature for five minutes or until lysate appears clear.
    6. Add 300μl neutralization Buffer 3.
    7. Mix thoroughly by inverting tube 8 times.
  • 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 50օC
  • 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.

Gibson Assembly

Step 1. - Reaction mix

Total Amount of Fragments Recommended Amount of Fragments Used for Assembly
2-3 Fragment Assembly 4-6 Fragment Assembly Positive Control**
0.02–0.5 pmols* (X μl) 0.2–1 pmols* (X μl) 10 μl
Gibson Assembly Master Mix (2X) 10 μl 10 μl 10 μl
Deionized H2O 10-X μl 10-X μl 0
Total Volume 20 μl 20 μl 10 μl

Step 2. - Incubation

We incubated our samples in a heat block at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Samples were kept on ice or at –20°C for subsequent transformation. If an assembly did not show good results, we repeated the protocol with extended incubation times as suggested on the NEB website.

Step 3. - Transformation

Samples were then used in 2ul aliquots to transform competent cells.

For full protocol click here

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.

    6. N.B. TBS is stable at 4°C for 3 mo.

Phosphate assay using media

Phosphate assay will work with media, however LB is not recommended due to high phosphate amount cause precipitation when tested. Instead 1x MOPS media, 4 mg/ml glucose and a known concentration of phosphate can be used. A recommend of 1mM phosphate can be used to get adequate cell growth without causing precipitation providing media is diluted by a 1in 20 dilution. Alternatively lower concentration can be used however growth will be affected. To see difference between samples, cells in exponential phase need to be captured and OD600 can be used to standardize against cell count. With MOPS 1mM phosphate an average time of 6 hours is recommend.

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 24 or 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

Manual growth assay

A manual growth assay can be conducted using a spectrometer at OD650. Samples are prepared in 50ml LB as (> 12hrs) in a 250ml conical flask, as MOPS media can be too slow. An initial start OD650 of 0.1 is used and then 1ml of sample can be removed from the conical flask hourly and optical density taken. These can then be plotted onto a growth curve and if desired sample can be detect from phosphate assay though for reliable media testing results MOPS would be recommended.