Difference between revisions of "Team:Glasgow/Protocols"

1. Dilute 400μl of overnight liquid culture into 20ml of broth with any necessary antibiotics to select for any plasmids already transformed into the cells
2. Incubate at 37⁰C, shaking at 225rpm for 90 minutes
3. Spin down for 2 minutes at 7000rpm at 4⁰C
4. Discard supernatant, resuspend pellet in 10ml of 50 mM CaCl₂, keep on ice
5. Repeat centrifugation for 2 minutes at 7000rpm at 4⁰C
6. Discard supernatant and the resuspend pellet in 1ml of 50 mM CaCl₂, keep on ice
7. CaCl₂ competent cells can be kept on ice in the fridge overnight

1. Add 1μl of plasmid DNA to 100μl of competent cells
2. Incubate on ice for 20 minutes
3. Heat shock at 42⁰C for 30 seconds or at 37⁰C for 5 minutes
4. Keep on ice for 2 minutes
5. Add 200μl of broth
6. Incubate the cells at 37⁰C. The time varies with the antibiotic resistance gene:

   Resistance	Time
   Chloramphenicol	90 minutes
   Kanamycin	60 minutes
   Ampicillin	30 minutes

7. Spread 200μl of transformed cells on plates with required antibiotic to select for plasmids
8. Incubate at 37°C overnight

1. Inoculate 400ml L-broth with 4ml culture
2. Incubate at 37⁰C, shaking at 250rpm until mid-log phase (OD600=0.5-0.7)
3. Split culture into 2 x 200ml samples
4. Chill on ice for 20 minutes
5. Spin 4000g for 15 minutes at 4⁰C
6. Re-suspend each in 200ml ice cold 10% glycerol
7. Spin 4000g for 15 minutes at 4⁰C
8. Re-suspend each in 100ml ice cold 10% glycerol
9. Spin 4000g for 15 minutes at 4⁰C
10. Re-suspend each in 10ml ice cold 10% glycerol
11. Spin 4000g for 15 minutes at 4⁰C
12. Re-suspend each in 500μl ice cold 10% glycerol
13. Aliquot 60μl into small eppendorf tubes and store at -70⁰C

1. Add 1μl of plasmid DNA to 30μl of competent cells
2. Leave on ice for 20 minutes
3. Transfer to pre-cooled electroporation cuvette
4. An electrical pulse was delivered by a Biorad Micropulser and 1ml L-broth immediately added
5. BCulture was then transferred to a 2ml nunc tube and incubated at 37⁰C for expression step (time varies dependent on antibiotic resistance gene in the plasmid that has just been transformed into the cells):

   Resistance	Time
   Chloramphenicol	90 minutes
   Kanamycin	60 minutes
   Ampicillin	30 minutes

6. Spread 100-200μl of transformed cells on dried L-agar plates with required antibiotic(s) to select for plasmid(s)
7. Incubate plates at 37°C overnight

1. A 20μl reaction typically contained:
   • 2μl of buffer
   • 4μl of miniprep (or 8μl G-Block) dependant on concentration of miniprep
   • Make up to 20μl with ddH20
   • 0.5-1.0μl of each restriction enzyme
2. Vortex
3. Incubate at 37⁰C for at least 60 minutes
4. Chill on ice for 20 minutes
5. Heat inactivate restriction enzymes

1. Pipette 1mL of bacterial overnight culture into a microcentrifuge tube
2. Centrifuge at 13,000 rpm for 1 min
3. Discard the supernatant
4. Repeat steps 1-3 two more times
5. Add 250μl of P1 buffer and pipette up and down to resuspend the pellet
6. Add 250μl of P2 buffer and invert Note: don’t allow this lysis reaction to proceed for more than 5 minutes
7. Add 350μl of N3 buffer to neutralise the reaction and invert
8. Centrifuge at 13,000 rpm for 10 minutes
9. Transfer 800μl of supernatant into a column
10. Centrifuge for 1 minute and discard flow-through
11. Add 500μl of PB buffer and centrifuge for 1 minute
12. Discard flow-through
13. Add 750μl of PE buffer and centrifuge for 1 minute
14. Discard flow-through
15. Centrifuge again for 1 minute to get rid of any residual buffer
16. Transfer the column to a microcentrifuge tube
17. Add 50μl of EB buffer and let it stand for 1 minute
18. Centrifuge for 1 minute

To make a 1L buffer:

1. Meaure 20mL of 50x TAE buffer
2. Transfer to a 2L measuring cylinder
3. Fill up to 1L with distilled water

To make a 1% agarose gel:

1. Weigh out 1g of agarose powder
2. Transfer to a microwave bottle
3. Pour 100mL of your buffer into the bottle
4. Microwave until clear
5. Cool dwn to 55⁰C before pouring the gel

To run the gel:

1. Place the gel in the tank and cover it with the buffer
2. Load 5μl of DNA ladder into the 2nd well (leave the first one empty)
3. Add 5μl of loading dye (30% glycerol, 1% bromophenol blue, 0.5% sodium dodecyl sulfate, diluted in TE buffer) into each restriction digest and pipette up and down a few times
4. Load 25μl of each restriction digest into a separate well
5. Run gel at 1.5 Amp, 100V for approximately 1 hour

To stain the gel:

• Ethidium bromide: stain in (concentration) for 40 minutes, destain for 40 minutes, image under UV light
• Azure A (for gel extraction): stain in 0.04%/20% ethanol for 15 minutes, destain for 15 minutes (multiple rounds of destaining may be required), image under visible light

1. Weigh an empty eppendorf and record the weight
2. Cut out a the band from the gel and place it in the eppendorf
3. Weigh the eppendorf again and calculate the weight difference
4. Add 3 volumes of Buffer QG to 1 volume of gel
5. Incubate at 50⁰C for 10 minutes until the agarose has dissolved. Vortex every 2-3 minutes
6. Add 1 volume of isopropanol and mix
7. Transfer the sample from the eppendorf to a QIAprep spin column in a 2mL collection tube
8. Centrifuge for 1 minute at 13,000rpm and discard flowthrough
9. Add 500μl of Buffer QG to column
10. Centrifuge for 1 minute at 13,000rpm and discard flowthrough
11. Add 750μl of Buffer PE and let it stand for 2-5 minutes
12. Centrifuge for 1 minute at 13,000rpm and discard flowthrough. Repeat this step
13. Transfer the column to a new eppendorf and discard the collection tube
14. Add 30μl of Buffer EB to the sample and let it stand for 1 minute
15. Centrifuge for 1 minute at 13,000rpm and keep discard the column

For a 10μl ligation reaction (these volumes change be changed depending on the concentration of the gel extracted DNA):

• 3μl of vector
• 5μl of insert
• 1μl of 10x ligation buffer
• 0.5μl of ddH₂O
• 0.5μl of ligase
• Vortex

For ligating oligo and vector:

• 1μl of oligo
• 5μl of vector
• 1μl of 10x ligation buffer
• 3μl of ddH₂O
• 0.5μl of ligase
• Vortex

1. Dissolve dried oligo in TE buffer to make a 100μM solution and leave for 10 minutes
2. To make a 100μl reaction:
• 10μl of top strand 100μM oligo
• 10μl of bottom strand 100μM oligo
• 80μl of TE buffer
• Heat at 86⁰C in a water bath for 5 minutes using a float
3. Scoop out some water and the float from the water bath using a large beaker
4. Let the water cool down slowly to approximately 30⁰C
5. Dilute annealed oligos 1/100

1. Prepare an overnight culture of S. thermophilus in M17L broth plus appropriate antibiotic
2. Prepare stock solution of P1 buffer, adding 20mg/ml lysozyme
3. Spin down 4-6ml of the overnight culture and resuspend pellet in 250μl of P1+lysozyme solution
4. Leave tube to incubate at 37⁰C for 30 minutes
5. Add 250μl of P2 buffer and invert Note: don’t allow this lysis reaction to proceed for more than 5 minutes
6. Add 350μl of N3 buffer to neutralise the reaction and invert
7. Centrifuge at 13,000 rpm for 10 minutes
8. Transfer 800μl of supernatant into a column
9. Centrifuge for 1 minute and discard flow-through
10. Add 500μl of PB buffer and centrifuge for 1 minute
11. Discard flow-through
12. Add 750μl of PE buffer and centrifuge for 1 minute
13. Discard flow-through
14. Centrifuge again for 1 minute to get rid of any residual buffer
15. Transfer the column to a microcentrifuge tube
16. Add 50μl of EB buffer and let it stand for 1 minute
17. Centrifuge for 1 minute

1. Prepare an overnight culture of S. thermophilus in M17L Broth in a static incubator at 37⁰C
2. Aliquot 1ml overnight culture into as many microfuge tubes as required for transformation
3. Centrifuge at 2000G for 9 minutes at room temperature
4. Discard supernatant, resuspend pellet in 1ml PBS, and repeat centrifugation. This should be carried out twice
5. Measure the OD600 and inoculate 1ml of milk at an OD of 0.05
6. Incubate for 1 hour at 37⁰C
7. Add 1μg DNA which S. thermophilus is to be transformed with, and 1μM of competence pheromone (ComS)
8. Incubate for 2.5 hours at 37⁰C
9. Spread on M17 plates containing antibiotic of choice (concentration of erythromycin used: 2.5μg/ml)

We used the RNeasy Mini Kit for RNA extraction from Qiagen.

A good supply of shortbread is essential to any productive lab environment. Recipe courtesy of BBC Food.

• 125g/4oz butter
• 55g/2oz caster sugar, plus extra to finish
• 180g/6oz plain flour

1. Heat the oven to 190⁰C/375F/Gas 5
2. Beat the butter and the sugar together until smooth.
3. Stir in the flour to get a smooth paste. Turn on to a work surface and gently roll out until the paste is 1cm/½in thick.
4. Cut into rounds or fingers and place onto a baking tray. Sprinkle with caster sugar and chill in the fridge for 20 minutes.
5. Bake in the oven for 15-20 minutes, or until pale golden-brown. Set aside to cool on a wire rack.