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<div class="accordion-item" data-accordion-item> | <div class="accordion-item" data-accordion-item> | ||
− | <a href="#" class="accordion-title">Preparation of | + | <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"> | <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>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> | ||
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<li>Discard supernatant and the resuspend pellet in 1ml of 50 mM CaCl2, keep on ice</li> | <li>Discard supernatant and the resuspend pellet in 1ml of 50 mM CaCl2, keep on ice</li> | ||
<li>CaCl2 competent cells can be kept on ice in the fridge overnight</li> | <li>CaCl2 competent cells can be kept on ice in the fridge overnight</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-item" data-accordion-item> | ||
+ | <a href="#" class="accordion-title">Transformation of CaCl<sub>2</sub> Competent Cells</a> | ||
+ | <div class="accordion-content" data-tab-content> | ||
+ | <ol class="protocol"> | ||
+ | <li>1μl of plasmid DNA was added to 100μl of competent cells</li> | ||
+ | <li>Samples were incubated on ice for 20 minutes</li> | ||
+ | <li>Heat shock was cried out at 37⁰C for 5 minutes</li> | ||
+ | <li>Cells were kept on ice for 2 minutes</li> | ||
+ | <li>200μl of broth was added and the cells incubated at 37⁰C for expression step (time varies dependent on antibiotic resistance gene in the plasmid that has just been transformed into the cells): | ||
+ | <ul> | ||
+ | <li>Chloramphenicol resistance = 90 minutes</li> | ||
+ | <li>Kanamycin resistance = 60 minutes</li> | ||
+ | <li>Ampicillin resistance = 30 minutes</li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li>100-200μl of transformed cells was spread on dried L-agar plates with required antibiotic(s) to select for plasmid(s) </li> | ||
+ | <li>Plates were incubated at 37°C overnight</li> | ||
</ol> | </ol> | ||
</div> | </div> |
Revision as of 13:59, 1 August 2016
Protocols
Here's the techniques we used blah blah blah"
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
- 1μl of plasmid DNA was added to 100μl of competent cells
- Samples were incubated on ice for 20 minutes
- Heat shock was cried out at 37⁰C for 5 minutes
- Cells were kept on ice for 2 minutes
- 200μl of broth was added and the cells incubated at 37⁰C for expression step (time varies dependent on antibiotic resistance gene in the plasmid that has just been transformed into the cells):
- Chloramphenicol resistance = 90 minutes
- Kanamycin resistance = 60 minutes
- Ampicillin resistance = 30 minutes
- 100-200μl of transformed cells was spread on dried L-agar plates with required antibiotic(s) to select for plasmid(s)
- Plates were incubated at 37°C overnight
Shortbread
A good supply of shortbread is essential to any productive lab environment.
- Heat the oven to 190C/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.
Protocol Two
Step One
Step Two
etc
Step Two
etc