Difference between revisions of "Team:Edinburgh UG/Experiments"
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<h2 style="text-align:left">LB Medium (1 litre liquid)</h2> | <h2 style="text-align:left">LB Medium (1 litre liquid)</h2> | ||
| − | < | + | <ul><li style="text-align:left">10 g tryptone</li> |
<li style="text-align:left">10 g NaCl</li> | <li style="text-align:left">10 g NaCl</li> | ||
<li style="text-align:left">5 g yeast extract</li> | <li style="text-align:left">5 g yeast extract</li> | ||
<li style="text-align:left">Water</li> | <li style="text-align:left">Water</li> | ||
| + | <li style="text-align:left">For selective medium, supplement with antibiotic as appropriate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin)</li> | ||
| + | </ul> | ||
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| + | <h2 style="text-align:left">LB Medium (1 litre solid / 50 plates)</h2> | ||
| + | <ul><li style="text-align:left">15 g agar agar</li> | ||
| + | <li style="text-align:left">10 g tryptone</li> | ||
| + | <li style="text-align:left">10 g NaCl</li> | ||
| + | <li style="text-align:left">5 g yeast extract</li> | ||
| + | <li style="text-align:left">Water</li> | ||
| + | <li style="text-align:left">For selective medium, supplement with antibiotic as appropriate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin)</li> | ||
| + | </ul> | ||
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| + | <section id="about" class="content-section text-center"> | ||
| + | <div class="container"> | ||
| + | <div class="row"> | ||
| + | <div class="col-lg-8 mx-auto"> | ||
| + | <h2 style="text-align:left">Chemically Competent Cells: CaCl2 Method</h2> | ||
| + | <ol><li style="text-align:left">Inoculate a single colony of appropriate cells into 10ml LB. Add antibiotic if needed and culture o/n at 37°C, 220rpm</li> | ||
| + | <li style="text-align:left">Inoculate 100ml LB with 1 ml o/n culture</li> | ||
| + | <li style="text-align:left">Incubate at 37 °C, 220rpm until OD600 = 0.3-0.6 (approx. 2 hrs)</li> | ||
| + | <li style="text-align:left">Transfer to 2 x 50 ml Falcon and leave on ice for 30 mins</li> | ||
| + | <li style="text-align:left">Centrifuge at 400 x g, 5 mins, 4 °C</li> | ||
| + | <li style="text-align:left">Resuspend pellet GENTLY in 25 ml ice cold 0.1 M MgCl2</li> | ||
| + | <li style="text-align:left">Incubate on ice for 30 min</li> | ||
| + | <li style="text-align:left">Centrifuge at 4000 xg, 5 min, 4 °C</li> | ||
| + | <li style="text-align:left">Resuspend pellet GENTLY in 25 ml ice cold 0.1 M CaCl2</li> | ||
| + | <li style="text-align:left">Incubate on ice for 30 min</li> | ||
| + | <li style="text-align:left">Centrifuge at 4000 xg, 5 min, 4 °C</li> | ||
| + | <li style="text-align:left">Resuspend pellet GENTLY in 1.25 ml ice cold CaCl2/Glycerol solution (1.7 ml 0.1 M CaCl2, 0.3 ml 100 % glycerol)</li> | ||
| + | <li style="text-align:left">Aliquot 100ul and flash freeze on dry ice. Store at – 80 °C</li> | ||
</ol> | </ol> | ||
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Revision as of 19:14, 14 October 2018
Experiments
General Protocols
Agarose Gel
- Weigh 1 g of agarose
- Add 100 mL of TAE 1X
- Heat up until the solution is homogeneous, avoiding boiling. If it boils, move away from the heat until it “calms down” and put it back on the heat until the agarose is completely dissolved
- While heating, prepare the bed in which the gel will polymerize. Make sure that it is well balanced and tight
- When homogeneous, add 10 µL of SYBR SAFE DNA Gel Stain to the solution and mix well
- Pour the solution into the bed and clear all its bubbles with a pipette tip. Add in the comb and make sure it is secure
- Mix the samples with loading dye in a 5:1 ratio. Put the samples into the wells, as well as 5 µL of DNA ladder into the first well
LB Medium (1 litre liquid)
- 10 g tryptone
- 10 g NaCl
- 5 g yeast extract
- Water
- For selective medium, supplement with antibiotic as appropriate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin)
LB Medium (1 litre solid / 50 plates)
- 15 g agar agar
- 10 g tryptone
- 10 g NaCl
- 5 g yeast extract
- Water
- For selective medium, supplement with antibiotic as appropriate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin)
Chemically Competent Cells: CaCl2 Method
- Inoculate a single colony of appropriate cells into 10ml LB. Add antibiotic if needed and culture o/n at 37°C, 220rpm
- Inoculate 100ml LB with 1 ml o/n culture
- Incubate at 37 °C, 220rpm until OD600 = 0.3-0.6 (approx. 2 hrs)
- Transfer to 2 x 50 ml Falcon and leave on ice for 30 mins
- Centrifuge at 400 x g, 5 mins, 4 °C
- Resuspend pellet GENTLY in 25 ml ice cold 0.1 M MgCl2
- Incubate on ice for 30 min
- Centrifuge at 4000 xg, 5 min, 4 °C
- Resuspend pellet GENTLY in 25 ml ice cold 0.1 M CaCl2
- Incubate on ice for 30 min
- Centrifuge at 4000 xg, 5 min, 4 °C
- Resuspend pellet GENTLY in 1.25 ml ice cold CaCl2/Glycerol solution (1.7 ml 0.1 M CaCl2, 0.3 ml 100 % glycerol)
- Aliquot 100ul and flash freeze on dry ice. Store at – 80 °C
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