# Protocols

### Making LB Agar plates

#### Preparation

Prepare liquid broth (LB) from liquid broth capsules and distilled water.

#### Making LB Agar (15%)

1. In a 1 litre bottle, add 3.75g of Agar powder to 250ml LB
2. Autoclave to sterilize
3. These can be stored and heated up to make plates when needed

#### Antibiotics

Different antibiotics will have different optimum concentrations

Chloramphenicol (25mg/ml)

Add 250mg to Chloramphnicol to 10ml ethanol (100%). Use 1ml for 1 litre of LB Agar to make final concentration 25ug/ml plates.

#### Making LB Agar Plates

1. Cool down/heat up LB agar to approx. 55 degrees Celsius
2. In a flow hood, add 250ul antibiotics to 250ml LB agar; mix
3. Pour approx. 25ml into each plate
4. Wait approx. 30 mins for plates to set
5. Store in 4 degrees Celsius fridge for future use

### Preparation of competent cells

• 1. Subculture an E.coli overnight culture 1:100 in LB (e.g. 500 μL overnight in 50 ml LB in a 250 ml flask). Incubate at 37°C with shaking to an OD600 of 0.375.
• Culture growth beyond OD 0.4 decreases transformation efficiency.
• 2. Aliquot 20 ml of the culture into chilled 50 ml tubes. Leave the tubes on ice for 5-10 minutes.
• Keep cells cold for all subsequent steps.
• 3. Centrifuge cells for 7 minutes at 1600 g, 4°C. Allow centrifuge to decelerate without brake.
• 4. Discard supernatant and resuspend each pellet in 4 ml ice cold ${\displaystyle CaCl2}$ solution.
• 5. Centrifuge cells for 5 minutes at 1100 g, 4°C.
• 6. Discard supernatant and resuspend each pellet in 4 ml ice cold CaCl2 solution. Keep on ice for 30 minutes.
• 7. Centrifuge cells for 5 minutes at 1100 g, 4°C.
• 8. Discard supernatant and resuspend each pellet in 800 μL ice cold CaCl2 solution.

It is important to resuspend this pellet well.

• 9. Aliquot 100 μL of this suspension into microcentrifuge tubes. Freeze in liquid nitrogen and store at -80°C.

### Miniprep Invitrogen Protocol

• 1.a. Inoculate the target bacteria in 1-5 mL medium broth (LB, YT o Terrific Broth) for 24 hours at 37°C. If the plasmid carries any marker, for example antibiotic resistance, add into the medium the marker substance.

Notes:

• Preheat an aliquot of TE Buffer (TE) to 65–70°C for eluting DNA. Heating is optional for eluting 1– 30 kb plasmid DNA but is recommended for eluting DNA >30 kb.

• Caution: Buffers contain hazardous reagents. Use caution when handling buffers.

• 1. Harvest. Centrifuge 1–5 mL of the overnight LB-culture. (Use 1–2 × 109 E. coli cells for each sample.) Remove all medium.
• 2. Resuspend. Add 250 μL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the pellet until it is homogeneous.
• 3. Lyse. Add 250 μL Lysis Buffer (L7). Mix gently by inverting the capped tube until the mixture is homogeneous. Do not vortex. Incubate the tube at room temperature for 5 minutes.
• 4. Precipitate. Add 350 μL Precipitation Buffer (N4). Mix immediately by inverting the tube, or for large pellets, vigorously shaking the tube, until the mixture is homogeneous. Do not vortex. Centrifuge the lysate at >12,000 × g for 10 minutes.
• 5. Bind. Attach the spin column with the supernatant from step 4 to a luer extension of the vacuum manifold. Apply vacuum. After all of the supernatant has passed through the column, turn off the vacuum.
• 6. Optional Wash. (Recommended for endA+ strains). Add 500 μL Wash Buffer (W10) with ethanol to the column. Incubate the column for 1 minute at room temperature. Apply vacuum. After all of the liquid has passed through the column, turn off the vacuum.
• 7. Wash. Add 700 μL Wash Buffer (W9) with ethanol to the column. Apply vacuum. After the liquid has passed through the column, turn off the vacuum.
• 8. Remove ethanol. Place the column into a 2-mL wash tube. Centrifuge the column at 12,000 × g for 1 minute. Discard the wash tube and flow-through.
• 9. Elute. Place the spin column in a clean 1.5-mL recovery tube. Add 75 μL of preheated TE Buffer (TE) to the center of the column. Incubate the column for 1 minute at room temperature.
• 10. Recover. Centrifuge the column at 12,000 × g for 2 minutes. The recovery tube contains the purified plasmid DNA. Discard the column. Store plasmid DNA at 4°C (short-term) or store the DNA in aliquots at −20°C (long-term).

### Heat Shock Transformation

#### Theory

Artificial transformation is a process whereby E.coli are made competent and take up DNA from their surroundings. The main steps are chilling the cells to 0oC with CaCl2 solution, adding the DNA and heat shocking the bacteria for a short period of time, allowing the cells to recover at 37oC and then plate them.

One hypothesis for artificial transformation is that the divalent cation provided by the chilled CaCl2 solution which is used in creating competent cells promotes an interaction between DNA and lipopolysaccharides which are also negatively charge. Lipopolysaccharides occur in higher densities near the parts of the outer membrane of Gram negative bacteria that are in close association with the inner membrane (zones of adhesion).

It is believed that prior to incubation DNA may interact with lipopolysaccharides and then cross the 'least barrier path' at the zones of adhesion described above.

#### Preparation

Ensure that you have enough sterilized liquid broth (or another medium such as SOC) and agar plates containing the correct antibiotic. Before you begin have a waterbath set to 42oC and an incubator at 37oC.

#### Practice

Take care not to disturb the competent E.coli: do not vortex them or pipette them up and down.

• Thaw competent E.coli cells ( 50 μl in an eppendorf tube, grown to an OD600 of 0.2 - 0.5) on ice. Let them sit on ice for at least 10 minutes.
• Add 1 μl of plasmid and mix gently by stirring with the pipette tip. Use more plasmid solution if it is very weak. We have had success with about 50 ng of plasmid DNA.
• Hold on ice for 0.5 hours
• Heat shock the cells with the DNA at 42ºC for 1 minute.
• Add 250 μl of SOC medium or LB broth

At this stage it is important to maintain sterility to avoid contaminating the liquid broth or the plates. Do not allow the pipette tip to touch anything before pipetting the liquid broth. Leaving the liquid broth bottle on the bench overnight should reveal any contamination in the morning.

• Incubate at 37ºC for 1 hour.
• Plate 10μl on agar containing an antibiotic (the antibiotic for which the plasmid confers resistance) and also plate 100μl on another identical plate. This allows for possible overcrowding so that single colonies can be selected.
• Incubate overnight at 37ºC.

Always keep agar plates upside down so that drips of condensation and falling debris does not contaminate them.

### BT transformation procedure

• 1) Inoculate the freshly-grown single colony of BT 4Q7 (on a nutrient agar plate) into 10 (or more 50-100 ml depend how much you cells you want) of BHI (Brain Heart Infusion) medium.
• 2) Let the culture grow for 12 hr at 30 ºC with shaking (250 rpm).
• 3) Incubate the culture on ice for 10 min
• 4) Harvest: 1,000 x g for 10 min at 4 ºC
• 5) Washing: 1,000 x g for 10 min (x 3 times) at 4 ºC using 10 ml of ice-cold sterilized ddw
• 6) Resuspend pellet in 1 ml of ice-cold 40% PEG 6000 (w/v)
• 7) Incubate the cells on ice for 10 min
• 8) Make 300 μl aliquot using 500 μl tubes
• 9) Add 2 to 5 μg of plasmid DNA into an aliquot and mix
• 10) Transfer the mixture into a 0.2 cm cuvette
• 11) Single pulse: 2.3 kV, 480 Ohm, 25 μF (¡ these are different that those used to transform E. coli!).
• 12) Add 3 ml of BHI medium into the mixture immediately
• 13) Transfer the whole thing into a 17 x 100 mm culture tube
• 14) Incubate the tube for an hour at 37 ºC with gentle shaking

15) Plate it out using BHI plates containing appropriate antibiotics 16) Usually colonies will appear in less than 24 hr.

PEG: Polietilenglicol 6000

### Gel Electrophoresis

• Gel preparation:
1. For 1% agarose gel (say 200ml), add 2g of agarose powder to 200 ml of 1x TAE buffer (obtained by diluting 10xTAE stock buffer)
• Note: The shorter the DNA strand lengths, the more concentrated the gel will be.
• Use 75-100ml of buffer for preparing one gel.
2. Heat the mixture in the microwave until the powder has completely dissolved stirring the contents every so often.
3. Transfer the solution into a disposable container.
4. Gel stains should be added when the agarose becomes cool enough to touch.(For SYBR Safe gel, add 5μl to 50ml TAE buffer)
• Electrophoresis setting:
1. Ensure electrophoresis chamber is clean and dry, tape the sides (with Autoclave tape, NOT standard masking tape) to make watertight. Slot in the desired comb.
2. Pipette a small amount of the tepid gel mixture around the edges of the taped regions to seal the chamber.
3. Add remaining gel solution to the chamber, and wait to set. The comb can then be removed from the chamber.
4. Fill the electrophoresis apparatus half-full with 1x TAE buffer solution (for good electrical contact) and place the set gel in the buffer. Ensure that there are no air bubbles (particularly in the wells created by the comb).
6. Connect the electrodes to the apparatus (the right way round!). Set DC voltage at 80V (with current at approximately 3 mA) and run for 30-60 minutes (or until the DNA has separated sufficiently).
• Tips for a Successful Gel:
• Add buffer, not water, when making the gel
• Seal the gel mould using autoclave tape (not masking tape) and with hot agarose
• After boiling buffer and agarose, let it cool before pouring into mould to prevent leakage
• Use running buffer to lubricate removal of mould else risk breaking the wells
• High salt is bad so dilute sample after enzymatic reactions
• Use full volume of well
• Check DNA is running towards the positive/cathode/red pole
• Check that your voltage and current are appropriate; running gel too fast will distort the bands
• Use fresh buffer for each gel, as a pH gradient will build up during each run

### Glycerol Stocks

Preparation of glycerol stocks of bacteria allows for long-term storage at -80°C without compromising viability of cells.

#### Theory

Freezing is an efficient way of storing bacteria. Glycerol allows to reduce the harmful effect of ice crystals of bacteria which can damage cells by dehydration caused by a localized increase in salt concentration leading to denaturation of proteins. Additionally, ice crystals can also puncture cellular membranes. Glycerol as a cryoprotectant depresses the freezing point of bacterial cells, enhancing supercooling. It does so by forming strong hydrogen bonds with water molecules, competing with water-water hydrogen bonding. This disrupts the crystal lattice formation of ice unless the temperature is significantly lowered.

#### Practice

• Preparation of glycerol stocks of bacteria
1. Prepare a liquid culture of bacteria in LB with antibiotic for selection. Use 100-fold dilution of an overnight culture of the strain of interest; or take bacterial colony from plate and inoculate in 5ml of LB.
2. Prepare sterile 60% glycerol by thoroughly mixing 60mL of 100% glycerol and 40mL D.I. Water; autoclave.
3. Incubate cells at 37° for 3-4 hours until the culture reaches the mid-log phase.
4. Transfer culture into an Eppendorf tube and mix with an appropriate amount of glycerol to get a final concentration of 20%.
5. For high viabilitystore at -80°.
• Tip:For precious strains, storage of two stock vials is recommended.
• Tip:Avoid repeated thawing and re-freezing of glycerol stocks as this can reduce the viability of the bacteria.
• Tip: When recovering a stored strain, it is advisable to check that the antibiotic markers have not been lost by streaking the strain onto an LB-agar plate containing the appropriate antibiotic(s).