(21 intermediate revisions by 4 users not shown) | |||
Line 9: | Line 9: | ||
<div class="protocols-block"> | <div class="protocols-block"> | ||
<div class="container"> | <div class="container"> | ||
− | <p> | + | <p>Described here are the various techniques we used in the course of our project</p> |
<div class="accordion" data-accordion data-multi-expand="true" data-allow-all-closed="true"> | <div class="accordion" data-accordion data-multi-expand="true" data-allow-all-closed="true"> | ||
<div class="accordion-item" data-accordion-item> | <div class="accordion-item" data-accordion-item> | ||
− | <a href="#" class="accordion-title"> | + | <a href="#" class="accordion-title">Preparation of CaCl<sub>2</sub> Competent Cells</a> |
<div class="accordion-content" data-tab-content> | <div class="accordion-content" data-tab-content> | ||
− | + | <ol class="protocol"> | |
− | + | <li>Dilute 400μl of overnight liquid culture into 20ml of broth with any necessary antibiotics to select for any plasmids already transformed into the cells </li> | |
− | + | <li>Incubate at 37⁰C, shaking at 225rpm for 90 minutes</li> | |
+ | <li>Spin down for 2 minutes at 7000rpm at 4⁰C</li> | ||
+ | <li>Discard supernatant, resuspend pellet in 10ml of 50 mM CaCl<sub>2</sub>, keep on ice</li> | ||
+ | <li>Repeat centrifugation for 2 minutes at 7000rpm at 4⁰C</li> | ||
+ | <li>Discard supernatant and the resuspend pellet in 1ml of 50 mM CaCl<sub>2</sub>, keep on ice</li> | ||
+ | <li>CaCl<sub>2</sub> competent cells can be kept on ice in the fridge overnight</li> | ||
+ | </ol> | ||
</div> | </div> | ||
</div> | </div> | ||
<div class="accordion-item" data-accordion-item> | <div class="accordion-item" data-accordion-item> | ||
− | <a href="#" class="accordion-title"> | + | <a href="#" class="accordion-title">Transformation of CaCl<sub>2</sub> Competent Cells</a> |
<div class="accordion-content" data-tab-content> | <div class="accordion-content" data-tab-content> | ||
− | + | <ol class="protocol"> | |
− | + | <li>Add 1μl of plasmid DNA to 100μl of competent cells</li> | |
− | + | <li>Incubate on ice for 20 minutes</li> | |
+ | <li>Heat shock at 42⁰C for 30 seconds <span class="i">or</span> at 37⁰C for 5 minutes</li> | ||
+ | <li>Keep on ice for 2 minutes</li> | ||
+ | <li>Add 200μl of broth | ||
+ | <li>Incubate the cells at 37⁰C. The time varies with the antibiotic resistance gene: | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Resistance</th> | ||
+ | <th>Time</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Chloramphenicol</td> | ||
+ | <td>90 minutes</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Kanamycin</td> | ||
+ | <td>60 minutes</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Ampicillin</td> | ||
+ | <td>30 minutes</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </li> | ||
+ | <li>Spread 200μl of transformed cells on plates with required antibiotic to select for plasmids</li> | ||
+ | <li>Incubate at 37°C overnight</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-item" data-accordion-item> | ||
+ | <a href="#" class="accordion-title">Preparation of Electrocompetent Cells</a> | ||
+ | <div class="accordion-content" data-tab-content> | ||
+ | <ol class="protocol"> | ||
+ | <li>Inoculate 400ml L-broth with 4ml culture</li> | ||
+ | <li>Incubate at 37⁰C, shaking at 250rpm until mid-log phase (OD600=0.5-0.7)</li> | ||
+ | <li>Split culture into 2 x 200ml samples</li> | ||
+ | <li>Chill on ice for 20 minutes</li> | ||
+ | <li>Spin 4000g for 15 minutes at 4⁰C</li> | ||
+ | <li>Re-suspend each in 200ml ice cold 10% glycerol</li> | ||
+ | <li>Spin 4000g for 15 minutes at 4⁰C</li> | ||
+ | <li>Re-suspend each in 100ml ice cold 10% glycerol</li> | ||
+ | <li>Spin 4000g for 15 minutes at 4⁰C</li> | ||
+ | <li>Re-suspend each in 10ml ice cold 10% glycerol</li> | ||
+ | <li>Spin 4000g for 15 minutes at 4⁰C</li> | ||
+ | <li>Re-suspend each in 500μl ice cold 10% glycerol</li> | ||
+ | <li>Aliquot 60μl into small eppendorf tubes and store at -70⁰C</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-item" data-accordion-item> | ||
+ | <a href="#" class="accordion-title">Transformation of Electrocompetent Cells</a> | ||
+ | <div class="accordion-content" data-tab-content> | ||
+ | <ol class="protocol"> | ||
+ | <li>Add 1μl of plasmid DNA to 30μl of competent cells</li> | ||
+ | <li>Leave on ice for 20 minutes</li> | ||
+ | <li>Transfer to pre-cooled electroporation cuvette</li> | ||
+ | <li>An electrical pulse was delivered by a Biorad Micropulser and 1ml L-broth immediately added</li> | ||
+ | <li>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): | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Resistance</th> | ||
+ | <th>Time</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Chloramphenicol</td> | ||
+ | <td>90 minutes</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Kanamycin</td> | ||
+ | <td>60 minutes</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Ampicillin</td> | ||
+ | <td>30 minutes</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </li> | ||
+ | <li>Spread 100-200μl of transformed cells on dried L-agar plates with required antibiotic(s) to select for plasmid(s)</li> | ||
+ | <li>Incubate plates at 37°C overnight</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-item" data-accordion-item> | ||
+ | <a href="#" class="accordion-title">Restriction Digests</a> | ||
+ | <div class="accordion-content" data-tab-content> | ||
+ | <ol class="protocol"> | ||
+ | <li>A 20μl reaction typically contained: | ||
+ | <ul> | ||
+ | <li>2μl of buffer</li> | ||
+ | <li>4μl of miniprep (or 8μl G-Block) dependant on concentration of miniprep</li> | ||
+ | <li>Make up to 20μl with ddH20</li> | ||
+ | <li>0.5-1.0μl of each restriction enzyme</li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li>Vortex</li> | ||
+ | <li>Incubate at 37⁰C for at least 60 minutes</li> | ||
+ | <li>Chill on ice for 20 minutes</li> | ||
+ | <li>Heat inactivate restriction enzymes</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-item" data-accordion-item> | ||
+ | <a href="#" class="accordion-title">Miniprep</a> | ||
+ | <div class="accordion-content" data-tab-content> | ||
+ | <ol class="protocol"> | ||
+ | <li>Pipette 1mL of bacterial overnight culture into a microcentrifuge tube</li> | ||
+ | <li>Centrifuge at 13,000 rpm for 1 min</li> | ||
+ | <li>Discard the supernatant</li> | ||
+ | <li>Repeat steps 1-3 two more times</li> | ||
+ | <li>Add 250μl of P1 buffer and pipette up and down to resuspend the pellet</li> | ||
+ | <li>Add 250μl of P2 buffer and invert <span class="i">Note: don’t allow this lysis reaction to proceed for more than 5 minutes</span></li> | ||
+ | <li>Add 350μl of N3 buffer to neutralise the reaction and invert</li> | ||
+ | <li>Centrifuge at 13,000 rpm for 10 minutes</li> | ||
+ | <li>Transfer 800μl of supernatant into a column</li> | ||
+ | <li>Centrifuge for 1 minute and discard flow-through</li> | ||
+ | <li>Add 500μl of PB buffer and centrifuge for 1 minute</li> | ||
+ | <li>Discard flow-through</li> | ||
+ | <li>Add 750μl of PE buffer and centrifuge for 1 minute</li> | ||
+ | <li>Discard flow-through</li> | ||
+ | <li>Centrifuge again for 1 minute to get rid of any residual buffer</li> | ||
+ | <li>Transfer the column to a microcentrifuge tube</li> | ||
+ | <li>Add 50μl of EB buffer and let it stand for 1 minute</li> | ||
+ | <li>Centrifuge for 1 minute</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-item" data-accordion-item> | ||
+ | <a href="#" class="accordion-title">Making a gel</a> | ||
+ | <div class="accordion-content" data-tab-content> | ||
+ | To make a 1L buffer: | ||
+ | <ol> | ||
+ | <li>Meaure 20mL of 50x TAE buffer</li> | ||
+ | <li>Transfer to a 2L measuring cylinder</li> | ||
+ | <li>Fill up to 1L with distilled water</li> | ||
+ | </ol><br> | ||
+ | To make a 1% agarose gel: | ||
+ | <ol> | ||
+ | <li>Weigh out 1g of agarose powder</li> | ||
+ | <li>Transfer to a microwave bottle</li> | ||
+ | <li>Pour 100mL of your buffer into the bottle</li> | ||
+ | <li>Microwave until clear</li> | ||
+ | <li>Cool dwn to 55⁰C before pouring the gel</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-item" data-accordion-item> | ||
+ | <a href="#" class="accordion-title">Gel electrophoresis</a> | ||
+ | <div class="accordion-content" data-tab-content> | ||
+ | To run the gel: | ||
+ | <ol class="protocol"> | ||
+ | <li>Place the gel in the tank and cover it with the buffer</li> | ||
+ | <li>Load 5μl of DNA ladder into the 2nd well (leave the first one empty)</li> | ||
+ | <li>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</li> | ||
+ | <li>Load 25μl of each restriction digest into a separate well</li> | ||
+ | <li>Run gel at 1.5 Amp, 100V for approximately 1 hour</li> | ||
+ | </ol> | ||
+ | To stain the gel: | ||
+ | <ul class="protocol"> | ||
+ | <li>Ethidium bromide: stain in (concentration) for 40 minutes, destain for 40 minutes, image under UV light</li> | ||
+ | <li>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</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-item" data-accordion-item> | ||
+ | <a href="#" class="accordion-title">Gel extraction using a Qiagen kit</a> | ||
+ | <div class="accordion-content" data-tab-content> | ||
+ | <ol class="protocol"> | ||
+ | <li>Weigh an empty eppendorf and record the weight</li> | ||
+ | <li>Cut out a the band from the gel and place it in the eppendorf</li> | ||
+ | <li>Weigh the eppendorf again and calculate the weight difference</li> | ||
+ | <li>Add 3 volumes of Buffer QG to 1 volume of gel</li> | ||
+ | <li>Incubate at 50⁰C for 10 minutes until the agarose has dissolved. Vortex every 2-3 minutes</li> | ||
+ | <li>Add 1 volume of isopropanol and mix</li> | ||
+ | <li>Transfer the sample from the eppendorf to a QIAprep spin column in a 2mL collection tube</li> | ||
+ | <li>Centrifuge for 1 minute at 13,000rpm and discard flowthrough</li> | ||
+ | <li>Add 500μl of Buffer QG to column</li> | ||
+ | <li>Centrifuge for 1 minute at 13,000rpm and discard flowthrough</li> | ||
+ | <li>Add 750μl of Buffer PE and let it stand for 2-5 minutes</li> | ||
+ | <li>Centrifuge for 1 minute at 13,000rpm and discard flowthrough. Repeat this step</li> | ||
+ | <li>Transfer the column to a new eppendorf and discard the collection tube</li> | ||
+ | <li>Add 30μl of Buffer EB to the sample and let it stand for 1 minute</li> | ||
+ | <li>Centrifuge for 1 minute at 13,000rpm and keep discard the column</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-item" data-accordion-item> | ||
+ | <a href="#" class="accordion-title">Ligation</a> | ||
+ | <div class="accordion-content" data-tab-content> | ||
+ | For a 10μl ligation reaction (these volumes change be changed depending on the concentration of the gel extracted DNA): | ||
+ | <ul> | ||
+ | <li>3μl of vector</li> | ||
+ | <li>5μl of insert</li> | ||
+ | <li>1μl of 10x ligation buffer</li> | ||
+ | <li>0.5μl of ddH<sub>2</sub>O</li> | ||
+ | <li>0.5μl of ligase</li> | ||
+ | <li>Vortex</li> | ||
+ | </ul> | ||
+ | For ligating oligo and vector: | ||
+ | <ul> | ||
+ | <li>1μl of oligo</li> | ||
+ | <li>5μl of vector</li> | ||
+ | <li>1μl of 10x ligation buffer</li> | ||
+ | <li>3μl of ddH<sub>2</sub>O</li> | ||
+ | <li>0.5μl of ligase</li> | ||
+ | <li>Vortex</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-item" data-accordion-item> | ||
+ | <a href="#" class="accordion-title">Annealing oligos</a> | ||
+ | <div class="accordion-content" data-tab-content> | ||
+ | <ol class="protocol"> | ||
+ | <li>Dissolve dried oligo in TE buffer to make a 100μM solution and leave for 10 minutes</li> | ||
+ | <li>To make a 100μl reaction:</li> | ||
+ | <ul> | ||
+ | <li>10μl of top strand 100μM oligo</li> | ||
+ | <li>10μl of bottom strand 100μM oligo</li> | ||
+ | <li>80μl of TE buffer</li> | ||
+ | <li>Heat at 86⁰C in a water bath for 5 minutes using a float</li> | ||
+ | </ul> | ||
+ | <li>Scoop out some water and the float from the water bath using a large beaker</li> | ||
+ | <li>Let the water cool down slowly to approximately 30⁰C</li> | ||
+ | <li>Dilute annealed oligos 1/100</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-item" data-accordion-item> | ||
+ | <a href="#" class="accordion-title"><span class="i">S. thermophilus</span> Miniprep protocol</a> | ||
+ | <div class="accordion-content" data-tab-content> | ||
+ | <ol class="protocol"> | ||
+ | <li>Prepare an overnight culture of <span class="i">S. thermophilus</span> in M17L broth plus appropriate antibiotic</li> | ||
+ | <li>Prepare stock solution of P1 buffer, adding 20mg/ml lysozyme</li> | ||
+ | <li>Spin down 4-6ml of the overnight culture and resuspend pellet in 250μl of P1+lysozyme solution</li> | ||
+ | <li>Leave tube to incubate at 37⁰C for 30 minutes</li> | ||
+ | <li>Add 250μl of P2 buffer and invert Note: don’t allow this lysis reaction to proceed for more than 5 minutes</li> | ||
+ | <li>Add 350μl of N3 buffer to neutralise the reaction and invert</li> | ||
+ | <li>Centrifuge at 13,000 rpm for 10 minutes</li> | ||
+ | <li>Transfer 800μl of supernatant into a column</li> | ||
+ | <li>Centrifuge for 1 minute and discard flow-through</li> | ||
+ | <li>Add 500μl of PB buffer and centrifuge for 1 minute</li> | ||
+ | <li>Discard flow-through</li> | ||
+ | <li>Add 750μl of PE buffer and centrifuge for 1 minute</li> | ||
+ | <li>Discard flow-through</li> | ||
+ | <li>Centrifuge again for 1 minute to get rid of any residual buffer</li> | ||
+ | <li>Transfer the column to a microcentrifuge tube</li> | ||
+ | <li>Add 50μl of EB buffer and let it stand for 1 minute</li> | ||
+ | <li>Centrifuge for 1 minute</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-item" data-accordion-item> | ||
+ | <a href="#" class="accordion-title"><span class="i">S. thermophilus</span> Transformation Protocol</a> | ||
+ | <div class="accordion-content" data-tab-content> | ||
+ | <ol class="protocol"> | ||
+ | <li>Prepare an overnight culture of S. thermophilus in M17L Broth in a static incubator at 37⁰C</li> | ||
+ | <li>Aliquot 1ml overnight culture into as many microfuge tubes as required for transformation</li> | ||
+ | <li>Centrifuge at 2000G for 9 minutes at room temperature</li> | ||
+ | <li>Discard supernatant, resuspend pellet in 1ml PBS, and repeat centrifugation. This should be carried out twice</li> | ||
+ | <li>Measure the OD600 and inoculate 1ml of milk at an OD of 0.05</li> | ||
+ | <li>Incubate for 1 hour at 37⁰C</li> | ||
+ | <li>Add 1μg DNA which S. thermophilus is to be transformed with, and 1μM of competence pheromone (ComS)</li> | ||
+ | <li>Incubate for 2.5 hours at 37⁰C</li> | ||
+ | <li>Spread on M17 plates containing antibiotic of choice (concentration of erythromycin used: 2.5μg/ml)</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-item" data-accordion-item> | ||
+ | <a href="#" class="accordion-title">RNA Extraction</a> | ||
+ | <div class="accordion-content" data-tab-content> | ||
+ | We used the RNeasy Mini Kit for RNA extraction from <a href="https://www.qiagen.com/gb/shop/sample-technologies/rna/rneasy-mini-kit/#orderinginformation"><b>Qiagen</b></a>. | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-item" data-accordion-item> | ||
+ | <a href="#" class="accordion-title">Shortbread</a> | ||
+ | <div class="accordion-content" data-tab-content> | ||
+ | A good supply of shortbread is essential to any productive lab environment. Recipe courtesy of <a href="http://www.bbc.co.uk/food/recipes/shortbread_1290">BBC Food</a>. | ||
+ | <ul class="ingredients"> | ||
+ | <li>125g/4oz butter</li> | ||
+ | <li>55g/2oz caster sugar, plus extra to finish</li> | ||
+ | <li>180g/6oz plain flour</li> | ||
+ | </ul> | ||
+ | <ol class="protocol"> | ||
+ | <li>Heat the oven to 190⁰C/375F/Gas 5</li> | ||
+ | <li>Beat the butter and the sugar together until smooth.</li> | ||
+ | <li>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.</li> | ||
+ | <li>Cut into rounds or fingers and place onto a baking tray. Sprinkle with caster sugar and chill in the fridge for 20 minutes.</li> | ||
+ | <li>Bake in the oven for 15-20 minutes, or until pale golden-brown. Set aside to cool on a wire rack.</li> | ||
+ | </ol> | ||
</div> | </div> | ||
</div> | </div> |
Latest revision as of 20:19, 19 October 2016
Protocols
Described here are the various techniques we used in the course of our project
Preparation of CaCl2 Competent Cells
- Dilute 400μl of overnight liquid culture into 20ml of broth with any necessary antibiotics to select for any plasmids already transformed into the cells
- Incubate at 37⁰C, shaking at 225rpm for 90 minutes
- Spin down for 2 minutes at 7000rpm at 4⁰C
- Discard supernatant, resuspend pellet in 10ml of 50 mM CaCl2, keep on ice
- Repeat centrifugation for 2 minutes at 7000rpm at 4⁰C
- Discard supernatant and the resuspend pellet in 1ml of 50 mM CaCl2, keep on ice
- CaCl2 competent cells can be kept on ice in the fridge overnight
Transformation of CaCl2 Competent Cells
- Add 1μl of plasmid DNA to 100μl of competent cells
- Incubate on ice for 20 minutes
- Heat shock at 42⁰C for 30 seconds or at 37⁰C for 5 minutes
- Keep on ice for 2 minutes
- Add 200μl of broth
- Incubate the cells at 37⁰C. The time varies with the antibiotic resistance gene:
Resistance Time Chloramphenicol 90 minutes Kanamycin 60 minutes Ampicillin 30 minutes - Spread 200μl of transformed cells on plates with required antibiotic to select for plasmids
- Incubate at 37°C overnight
Preparation of Electrocompetent Cells
- Inoculate 400ml L-broth with 4ml culture
- Incubate at 37⁰C, shaking at 250rpm until mid-log phase (OD600=0.5-0.7)
- Split culture into 2 x 200ml samples
- Chill on ice for 20 minutes
- Spin 4000g for 15 minutes at 4⁰C
- Re-suspend each in 200ml ice cold 10% glycerol
- Spin 4000g for 15 minutes at 4⁰C
- Re-suspend each in 100ml ice cold 10% glycerol
- Spin 4000g for 15 minutes at 4⁰C
- Re-suspend each in 10ml ice cold 10% glycerol
- Spin 4000g for 15 minutes at 4⁰C
- Re-suspend each in 500μl ice cold 10% glycerol
- Aliquot 60μl into small eppendorf tubes and store at -70⁰C
Transformation of Electrocompetent Cells
- Add 1μl of plasmid DNA to 30μl of competent cells
- Leave on ice for 20 minutes
- Transfer to pre-cooled electroporation cuvette
- An electrical pulse was delivered by a Biorad Micropulser and 1ml L-broth immediately added
- 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 - Spread 100-200μl of transformed cells on dried L-agar plates with required antibiotic(s) to select for plasmid(s)
- Incubate plates at 37°C overnight
Restriction Digests
- 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
- Vortex
- Incubate at 37⁰C for at least 60 minutes
- Chill on ice for 20 minutes
- Heat inactivate restriction enzymes
Miniprep
- Pipette 1mL of bacterial overnight culture into a microcentrifuge tube
- Centrifuge at 13,000 rpm for 1 min
- Discard the supernatant
- Repeat steps 1-3 two more times
- Add 250μl of P1 buffer and pipette up and down to resuspend the pellet
- Add 250μl of P2 buffer and invert Note: don’t allow this lysis reaction to proceed for more than 5 minutes
- Add 350μl of N3 buffer to neutralise the reaction and invert
- Centrifuge at 13,000 rpm for 10 minutes
- Transfer 800μl of supernatant into a column
- Centrifuge for 1 minute and discard flow-through
- Add 500μl of PB buffer and centrifuge for 1 minute
- Discard flow-through
- Add 750μl of PE buffer and centrifuge for 1 minute
- Discard flow-through
- Centrifuge again for 1 minute to get rid of any residual buffer
- Transfer the column to a microcentrifuge tube
- Add 50μl of EB buffer and let it stand for 1 minute
- Centrifuge for 1 minute
Making a gel
To make a 1L buffer:
To make a 1% agarose gel:
- Meaure 20mL of 50x TAE buffer
- Transfer to a 2L measuring cylinder
- Fill up to 1L with distilled water
To make a 1% agarose gel:
- Weigh out 1g of agarose powder
- Transfer to a microwave bottle
- Pour 100mL of your buffer into the bottle
- Microwave until clear
- Cool dwn to 55⁰C before pouring the gel
Gel electrophoresis
To run the gel:
- Place the gel in the tank and cover it with the buffer
- Load 5μl of DNA ladder into the 2nd well (leave the first one empty)
- 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
- Load 25μl of each restriction digest into a separate well
- Run gel at 1.5 Amp, 100V for approximately 1 hour
- 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
Gel extraction using a Qiagen kit
- Weigh an empty eppendorf and record the weight
- Cut out a the band from the gel and place it in the eppendorf
- Weigh the eppendorf again and calculate the weight difference
- Add 3 volumes of Buffer QG to 1 volume of gel
- Incubate at 50⁰C for 10 minutes until the agarose has dissolved. Vortex every 2-3 minutes
- Add 1 volume of isopropanol and mix
- Transfer the sample from the eppendorf to a QIAprep spin column in a 2mL collection tube
- Centrifuge for 1 minute at 13,000rpm and discard flowthrough
- Add 500μl of Buffer QG to column
- Centrifuge for 1 minute at 13,000rpm and discard flowthrough
- Add 750μl of Buffer PE and let it stand for 2-5 minutes
- Centrifuge for 1 minute at 13,000rpm and discard flowthrough. Repeat this step
- Transfer the column to a new eppendorf and discard the collection tube
- Add 30μl of Buffer EB to the sample and let it stand for 1 minute
- Centrifuge for 1 minute at 13,000rpm and keep discard the column
Ligation
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
- 1μl of oligo
- 5μl of vector
- 1μl of 10x ligation buffer
- 3μl of ddH2O
- 0.5μl of ligase
- Vortex
Annealing oligos
- Dissolve dried oligo in TE buffer to make a 100μM solution and leave for 10 minutes
- 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
- Scoop out some water and the float from the water bath using a large beaker
- Let the water cool down slowly to approximately 30⁰C
- Dilute annealed oligos 1/100
S. thermophilus Miniprep protocol
- Prepare an overnight culture of S. thermophilus in M17L broth plus appropriate antibiotic
- Prepare stock solution of P1 buffer, adding 20mg/ml lysozyme
- Spin down 4-6ml of the overnight culture and resuspend pellet in 250μl of P1+lysozyme solution
- Leave tube to incubate at 37⁰C for 30 minutes
- Add 250μl of P2 buffer and invert Note: don’t allow this lysis reaction to proceed for more than 5 minutes
- Add 350μl of N3 buffer to neutralise the reaction and invert
- Centrifuge at 13,000 rpm for 10 minutes
- Transfer 800μl of supernatant into a column
- Centrifuge for 1 minute and discard flow-through
- Add 500μl of PB buffer and centrifuge for 1 minute
- Discard flow-through
- Add 750μl of PE buffer and centrifuge for 1 minute
- Discard flow-through
- Centrifuge again for 1 minute to get rid of any residual buffer
- Transfer the column to a microcentrifuge tube
- Add 50μl of EB buffer and let it stand for 1 minute
- Centrifuge for 1 minute
S. thermophilus Transformation Protocol
- Prepare an overnight culture of S. thermophilus in M17L Broth in a static incubator at 37⁰C
- Aliquot 1ml overnight culture into as many microfuge tubes as required for transformation
- Centrifuge at 2000G for 9 minutes at room temperature
- Discard supernatant, resuspend pellet in 1ml PBS, and repeat centrifugation. This should be carried out twice
- Measure the OD600 and inoculate 1ml of milk at an OD of 0.05
- Incubate for 1 hour at 37⁰C
- Add 1μg DNA which S. thermophilus is to be transformed with, and 1μM of competence pheromone (ComS)
- Incubate for 2.5 hours at 37⁰C
- Spread on M17 plates containing antibiotic of choice (concentration of erythromycin used: 2.5μg/ml)
RNA Extraction
We used the RNeasy Mini Kit for RNA extraction from Qiagen.
Shortbread
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
- Heat the oven to 190⁰C/375F/Gas 5
- Beat the butter and the sugar together until smooth.
- 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.
- Cut into rounds or fingers and place onto a baking tray. Sprinkle with caster sugar and chill in the fridge for 20 minutes.
- Bake in the oven for 15-20 minutes, or until pale golden-brown. Set aside to cool on a wire rack.