Team:Newcastle/Notebook/protocols

Contents 1 Streak plate 1.1 From glycerol stocks 1.2 From liquid culture 2 Liquid culture 3 Glycerol stocks 3.1 Making glycerol stocks 4 Chromosomal DNA Extraction 4.1 Nanodrop quantification of DNA extracts 5 PCR 6 QIAprep Spin Miniprep Kit 7 Making a culture of competent Bacillus subtilis 168 7.1 Preparing SMM media 7.2 Preparing MM competence media 7.3 Preparing starvation media 8 Production of L-form containing Plants 9 Visualisation of L-forms in plants 10 L-form Media Components 11 L-form Growth Media 11.1 Solid Media 11.2 Liquid media 12 Creating L-forms 13 Lysozyme Protoplast Formation 14 Making Starch Agar Plates 15 L-form Media Components 15.1 2×MSM 15.2 2×NB 15.3 2×NA 15.4 Stock PenG 15.5 Solid Media for L-form growth 15.6 Liquid Media for L-form growth

Streak plate

From glycerol stocks

1. Glycerol stock must be kept in an ice bucket.
2. Working close to lit Bunsen burner.
3. Flame a loop until red hot then dip into glycerol stock in cryo-vial.
4. Streak plate by: -
   1. Dragging loop across agar 5-10 times.
   2. Flame loop, then dip in agar (away from streak) to cool.
   3. Streak 4 times at a 90o angle from first streak.
   4. Streak again 4 times at 90o from second set of streaks.
   5. Streak again 4 times at 90o from third set of streaks to complete a square on the agar plate.
   6. Finally drag loop in a squiggle motion across the centre of the agar plate.
   7. The streak plate must then be left overnight at 37oC for colonies to grow.
   8. LB agar plates will usually be used unless alternative conditions are required.

From liquid culture

Protocol is similar to that of creating streak plates from glycerol stocks. However, an overnight culture of the strain to be streaked is grown and the loop is dipped into this culture rather than a cryo-vial of the glycerol stock.  

Liquid culture

Working close to lit Bunsen burner:

1. Liquid cultures can be grown in plastic universal tubes if only a small volume is needed (e.g. 5ml).
2. Transfer a colony from a plate of colonies using a flame-sterilised loop into liquid LB media in tube.
3. Cultures should be made from only one colony.
4. Leave the plastic universals on rotating plate (aerator) at 37°C overnight.

 

Glycerol stocks

Making glycerol stocks

NB: Only 50% glycerol solution is necessary for preserving cells by freezing at -80oC.

1. 1 volume of 100% glycerol should be mixed with 1 volume of Mili-Q filtered water (i.e. 1ml glycerol for 1ml water).
2. Vortex the solution briefly to mix.
3. Filter the solution using a sterile syringe filter, then pipette 500µl of the 50% glycerol solution into cryo-vials.
4. Pipette 500μl of the LB cell culture to be preserved into the cryo-vials.
5. Mix by inverting the tubes.
6. Glycerol stocks can then be stored in deep freeze at -80oC.

 

Chromosomal DNA Extraction

1. Add 1.75ml of bacterial cell culture to a 2ml tube.
2. Spin tubes at 20,000 x g (max speed of centrifuge may be 16,000 x g, if so spin at this speed) for 5 minutes in centrifuge and then discard the liquid supernatant.
3. Add 180μl of enzymatic lysis buffer (36μl lysozyme and 144μl buffer) to the tube and vortex for 10-20 seconds.
4. Incubate at 37oC for 20 minutes.
5. Add 25μl proteinase K.
6. Add 200μl Buffer AL.
7. Vortex briefly.
8. Incubate at 56oC for 5 minutes.
9. Add 200μl ethanol to the tube and vortex briefly.
10. Transfer entire contents (~600μl) to a spin column in 2ml collection tube using a pipette.
11. Centrifuge column at 10,000 x g for 1 minute.
12. Discard flow through and replace spin column in a 2ml collection tube.
13. Add 500μl Buffer AW1 to the column and centrifuge at 10,000 x g for 1 minute.
14. Discard flow through and replace spin column in a 2ml collection tube.
15. Add 500μl Buffer AW2 to the column and centrifuge at 16,000 x g for 3 minutes.
16. Carefully remove tubes from centrifuge without allowing the flow-through to contact the column (if this happens, spin tube in centrifuge again for 1 minute at 16,000 x g) and transfer column to a 1.5ml tube.
17. Add 200μl Buffer AE to the column and leave to stand at room temperature for 1 minute.
18. Centrifuge at 10,000 x g for 1 minute and then discard the column. Store the DNA in the collection tube at 4oC.

NB: Steps 17 and 18 can be carried out in repeat using smaller volumes of Buffer AE in order to try and increase DNA yield. Adding 30µl Buffer AE to the column and leaving to stand at room temperature for 5 minutes before centrifuging (and repeating 4-6 times) produces a high DNA yield.

 

Nanodrop quantification of DNA extracts

Nanodrop procedure:

1. Log into computer.
2. Open Nanodrop 1000.
3. Click on ‘Nucleic Acids’ button.
4. Default DNA setting will be DNA-50 – this is correct.
5. Wipe pedestal and top with tissue paper before starting.
6. Initialise by adding 3µl H2O to pedestal as instructed by the program.
7. Blank with 3µl elution Buffer (EB) (click ‘Blank’).
8. Add 3µl sample and click ‘Measure’.
9. Wipe pedestal with tissue paper between measurements.
10. Re-blank with water when finished to clean instrument, dry with tissue and log off.

PCR

1. Add the following reagents (quantities are for a 50µl reaction) to a PCR tube in the listed order:
   1. 27.5µl H2O
   2. 10.0µl 5x Buffer HF
   3. 1.0µl dNTP (200 µM)
   4. 5.0µl Reverse primer
   5. 5.0µl Forward primer
   6. 1.0µl Template DNA
   7. 0.5µl Phusion Polymerase (must be added last)
2. Mix these reagents by inverting the tube and then centrifuge for a couple of seconds for all of the reagents to collect at the bottom of the tube.
3. Set conditions of PCR on PCR machine:
   • 98°C 30 seconds initial denaturation
   • 98°C 10 seconds denaturation
   • x°C (10-20 seconds) anneal
   • 72°C 30 seconds/kb extension
   • 72°C 5-10 minutes final extension
   • 4°C hold
4. Store the PCR products at 4oC.

NB: The denaturation, anneal and extension steps are repeated cyclically for 30 cycles.

NB: Procedure for PCR differs between DNA polymerases. This procedure is for Phusion DNA polymerase.

QIAprep Spin Miniprep Kit

1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
2. Add 250 µl Buffer P2and mix by inverting the tube 4-6 times.
3. Add 350 µl Buffer N3 and mix immediately by inverting 4-6 times.
4. Centrifuge for 10 min at 13,000 rpm in a table top microcentrifuge.
5. Apply the supernatant to the QIAprep spin column by pipetting.
6. Centrifuge for 30-60s. Discard the flow-through.
7. Wash the QIAprep spin column by adding 0.5ml Buffer PB and centrifuging for 30-60s. Discard the flow-through.
8. Wash QIAprep spin column by adding 0.75ml Buffer PE and centrifuging for 30-60s.
9. Discard the flow-through, and centrifuge for an additional min to remove residual wash buffer.
10. To elute the DNA, put the QIAprep column in a clean 1.5ml microcentrifuge tube and add 50 µl Buffer EB to the centre of each QIAprep spin column. Let it stand for 1 min and centrifuge for 1 min.

Making a culture of competent Bacillus subtilis 168

1. Inoculate 5ml of MM competence media with a single colony off the plate containing the strain in a 15ml falcon tube and incubate at 37°C and 180 rpm overnight
2. Next day, transfer 0.3ml of overnight culture into fresh 5ml MM competence media in a 50ml falcon tube and incubate for 3 hours at 37°C and 180 rpm
3. Add 5ml of pre-warmed starvation medium and incubate for a further 2 hours at 37°C and 180 rpm
4. Transfer 0.4ml of the culture to a 1.5ml microfuge tube and add 10µl DNA
5. Incubate for 1 hour at 37°C and 180 rpm – place tubes on sides to ensure maximum aeration
6. Plate out 200µl with appropriate antibiotic incubate over night at 37°C and 180 rpm

Preparing SMM media

For 1 litre you need:

• 2.0g ammonium sulphate
• 14.0g dipotassium hydrogen phosphate
• 6.0g potassium dihydrogen phosphate
• 1.0g sodium citrate dehydrate (trisodium citrate)
• 0.2g magnesium sulphate

Make it up to 1 litre, split into 5X 200ml bottles and autoclave

Preparing MM competence media

For 5ml you need:

• 5ml SMM media
• 62.5µl solution E (40% glucose)
• 50µl Tryptophan solution
• 30µl solution F
• 5µl casamino acids
• 2.5µl Fe-NH4-citrate

Preparing starvation media

For 5ml you need:

• 5ml SMM media
• 62.5µl solution E (40% glucose)
• 30µl solution F (MgSO4)

Production of L-form containing Plants

1. Rinse seeds for 2 minutes in 70% (v/v) ethanol.
2. Soak seeds for 10 minutes in 20% (v/v) Milton’s sterilising fluid (0.4% (v/v) NaOCl final concentration).
3. Wash seeds thoroughly five times in sterile distilled water and leave in final wash for fifteen minutes.
4. Place seeds in (9cm) Petri dishes (20-25 seeds per dish) containing Murashige and Skoog (M and S) basal medium, solidified with 0.8% (w/v) agar no.1 (Oxoid, UK) and incubate at 25 degrees Celsius in the dark until radicals just appear (this should take 21-24hr for Chinese cabbage).
5. Harvest transformed L-forms from 3-4 day old LPM plates using 5% (w/v) mannitol into sterile plastic universal tubes.
6. Use spectrophotometry (OD600) to produce a bacterial suspension containing approximately 107 CFU ml-1.
7. Select seeds with radicals 1-2mm in length and soak in the bacterial suspension (20 seeds per 10ml) for 3 hours at 25 degrees Celsius, gently shaking the seeds by hand every 30 minutes. Treat some seedlings with 5% (w/v) mannitol instead of L-forms to act as a control.
8. Wash seeds ten times in distilled water to lyse any extracellular L-forms.
9. Place plants in individual magenta pots with 50ml M and S basal medium solidified with 0.8% (w/v) agar no. 1.
10. Incubate seeds, in a plant growth chamber (if available), with 16 hour light regime (300-400 µE m-2s-1) at 25 degrees Celsius.

Visualisation of L-forms in plants

For the visualisation of L-forms in plants, both of the following solutions must be produced prior to commencing the procedure.

Gus staining solution

• 0•5 mg ml−1 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, X-gluc (Sigma)
• 100 mM sodium phosphate buffer (pH 7•0)
• 1% Triton X-100
• 1%N,N′ dimethylformamide (DMF)
• 10 mM EDTA

Fixing solution

• 5% formaldehyde (v/v)
• 5% (v/v) acetic acid
• 20% (v/v) ethanol

1. Harvest batches of five seedlings at daily intervals for seven days after treatment.
2. Vacuum-infiltrate intact seedlings in a vacuum oven for 20 with gus staining solution.
3. Cover the infiltrated seedlings and incubate in staining solution at 37 degrees Celsius until blue colour appears (approximatly 2 hours).
4. Wash seedlings twice with 50% (v/v) glycerol to remove gus staining solution and re-suspend in 50% (v/v) glycerol.
5. When blue colour appears, fix in fixing solution.
6. Store in 100% ethanol.

L-form Media Components

PROTOCOL TO COME

L-form Growth Media

Solid Media

PROTOCOL TO COME

Liquid media

PROTOCOL TO COME

Creating L-forms

PROTOCOL TO COME

Lysozyme Protoplast Formation

Making Starch Agar Plates

• Add 1g starch powder (to 100mL LB)

• Add 1g bacterial agar powder

• Mix by shaking gently and microwave until solute has completely dissolved

• Pour into pre-labelled petri-dishes (100mL for 4 plate)

• Remove any bubbles by quickly moving the flame of a Bunsen burner over the poured agar

• Leave to set 30-45 minutes

• Put the plates in a fan-cupboard for 10 minutes with their lids partially off to dry out any condensation before plating

L-form Media Components

2×MSM

Two times concentrated stock of 1 M sucrose 40 mM Maleic acid 40 mM Magnesium Chloride.

• 342 g Sucrose
• 4.65 g Maleic acid
• 8.12 g Magnesium Chloride

1. Add d-H2O to a final volume of 1 l
2. Adjust pH 7 with NaOH
3. Aliquot into 500 ml bottles
4. Autoclave on a short cycle (15 minutes at 121°C) as prolonged heating caramelizes the sugar giving a dark brown solution that the bacteria don’t grow in.

2×NB

Add 2.6 g of NB to 100 ml of demin-H2O then autoclave.

2×NA

Add 5.6 g of NA to 100 ml of d-H2O then autoclave.

Stock PenG

200 mg/ml of Penicillin G (benzylpenicillin or PenG, Sigma)

Add 2 g of PenG to 10 ml final volume of d-H2O and filter sterilise. Aliquot and freeze at -20°C.

Solid Media for L-form growth

1. First make 2 times concentrated Nutrient agar (Oxoid, NA), 2 times concentrated MSM and 200 mg/ml.
2. Allow the nutrient agar to cool to about 80-90°C
3. Add an equal volume room temperature MSM.
4. Mix thoroughly.
5. Add 200 µL of 200 mg/ml PenG and pour.
6. Allow the agar to set and cool to room temperature
7. Air dry for no more than 5 minutes.

N.B. The last two steps are critical as drying the plates leads to evaporation of water which increases the concentration of sucrose: this should be avoided as the L-forms could be sensitive to changes in the osmolarity of the media.

Liquid Media for L-form growth

1. Mix equal volumes of 2×MSM and 2×Nutrient Browth (Oxoid, NB) and add PenG if required.
2. Add 14-20 mL of media to a 250 ml Erlenmeyer flask. Or 8-10 ml into 100 ml flask (to allow a large interface surface with air that will ensure proper oxygenation)
3. Suck up 5-10 µL of cells the sticky secretion from an L-form colony with a pipette and inoculate the media.
4. Gently mix the media to break up the cells
5. Incubate at 30°C without shaking.

N.B. A large media volume to flask volume ratio allows a thin layer of media which ensures diffusion of oxygen. Shaking is not necessary for growth, and if done should be very gently.