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Team:Queens Canada/Notebook/Protocols/HeatShock
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<span class="classred"><a href="https://2011.igem.org/Team:Queens_Canada/Notebook/Protocols/Extraction"> extraction </a></span> </regulartext> | <span class="classred"><a href="https://2011.igem.org/Team:Queens_Canada/Notebook/Protocols/Extraction"> extraction </a></span> </regulartext> | ||
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| + | <h3red> Heat Shock (Transformation) </h3red> <p> | ||
| - | + | <regulartext> <b> Storage and Labelling </b> </regulartext> <br> | |
| + | <regulartext> - Store the transformed cells in the 4°C fridge. </regulartext> <br> | ||
| + | <regulartext> - Label the product plate as “EC (resistance)” and in accordance with the standard labelling format, as outlined at the front of your lab book and on Google Docs. </regulartext> <p> | ||
| + | <regulartext> <b> Materials </b> </regulartext> <br> | ||
| + | <regulartext> - 100μL of chemical competent cells per transformation </regulartext> <br> | ||
| + | <regulartext> - Miniprep plasmid DNA </regulartext> <br> | ||
| + | <regulartext> -2XTY or SOC medium </regulartext> <br> | ||
| + | <regulartext> - Antibiotic plates (according to plasmid) </regulartext> <p> | ||
| + | <regulartext> <b> Procedure </b> </regulartext> <br> | ||
| + | <regulartext> 1. Take out competent cells from -80⁰C and put them on ice immediately before they are needed. <br> | ||
| + | 2. Add 2μl plasmid DNA to thawed cells and mix by flicking the side of the tube. <br> | ||
| + | 3. Incubate on ice for 20 minutes. <br> | ||
| + | 4. Pre-warm antibiotic plates in 37⁰C incubator. <br> | ||
| + | 5. Heat shock for 1 min 15 sec at 42⁰C.<br> | ||
| + | 6. Place on Ice for 2 minutes. <br> | ||
| + | 7. Add 500ul 2XTY (or SOC) medium (kept at room temp) to each tube (medium with NO antibiotic). <br> | ||
| + | 8. Shake the tubes at 37⁰C for 1 hour on a shaking incubator. <br> | ||
| + | 9. Spread 100μl of each transformation tube on appropriate antibiotic plates. <br> | ||
| + | -In addition, you can: Take 1μL of TOP10 and add 99μL of growth buffer, plate it. Spin remainder for 1 min at 13000, discard supernatant, re-suspend in 100μL of growth buffer<br> | ||
| + | 10. Incubate at 37⁰C overnight.<br> | ||
| + | </regulartext> <br> | ||
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Latest revision as of 20:32, 28 September 2011
2. Add 2μl plasmid DNA to thawed cells and mix by flicking the side of the tube.
3. Incubate on ice for 20 minutes.
4. Pre-warm antibiotic plates in 37⁰C incubator.
5. Heat shock for 1 min 15 sec at 42⁰C.
6. Place on Ice for 2 minutes.
7. Add 500ul 2XTY (or SOC) medium (kept at room temp) to each tube (medium with NO antibiotic).
8. Shake the tubes at 37⁰C for 1 hour on a shaking incubator.
9. Spread 100μl of each transformation tube on appropriate antibiotic plates.
-In addition, you can: Take 1μL of TOP10 and add 99μL of growth buffer, plate it. Spin remainder for 1 min at 13000, discard supernatant, re-suspend in 100μL of growth buffer
10. Incubate at 37⁰C overnight.
