Difference between revisions of "Team:BOKU-Vienna/Protocol"
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<p> • Frozen chemically competent cells (100 µL per tube) are thawed on ice. <br> • The entire ligation (or other DNA sample to be transformed) is added to the cells, and the mix incubated on ice for 30 minutes. <br> • The cells and DNA mix is heat shocked for 90 s at 42 °C. <br> •Allow the cells to recover on ice for 5 minutes. <br> • Add 1 mL of LB medium to the cells and incubate the tube at 37 °C in a shaker-incubator (150 rpm) for 45 min to 1h. <br> • Plate the appropriate amount of cells on selective LB agar.</p> | <p> • Frozen chemically competent cells (100 µL per tube) are thawed on ice. <br> • The entire ligation (or other DNA sample to be transformed) is added to the cells, and the mix incubated on ice for 30 minutes. <br> • The cells and DNA mix is heat shocked for 90 s at 42 °C. <br> •Allow the cells to recover on ice for 5 minutes. <br> • Add 1 mL of LB medium to the cells and incubate the tube at 37 °C in a shaker-incubator (150 rpm) for 45 min to 1h. <br> • Plate the appropriate amount of cells on selective LB agar.</p> | ||
| − | + | <h4 id="miniprep" class="test" >Plasmid DNA extraction</h4><br> | |
</div> | </div> | ||
Revision as of 13:37, 15 September 2017
Protocol
Materials
LB medium
| Name | Amount |
|---|---|
| Peptone | 10 g |
| Yeast Extract | 5 g |
| NaCl | 5 g |
| RO-water | 1000 mL |
LB agar
| Name | Amount |
|---|---|
| Peptone | 10 g |
| Yeast Extract | 5 g |
| NaCl | 5 g |
| RO water | 986 mL |
| Agar | 14 g |
Methods
Preparation of chemical competent E. coli
• E. coli strain DH10B is inoculated from a glycerol stock onto an LB plate; the inoculum is streaked on the plate using a loop so as to obtain individual colonies. The plate is incubated overnight at 37 °C.
• Inoculate 10 mL of LB medium with a single colony and incubate the flask overnight in a shaker-incubator (37°C, shaking 180 rpm).
• The following day, transfer 2 mL of this culture to a flask containing 200 mL LB medium and incubate for about 2h until OD600 raches 0.6.
• Cool down the cells on ice for 10 minutes. The cells are pelleted in a centrifuge for 5 min at 4,500 rpm (4,000 x g) at 4°C. The cells are resuspended in 0.4 volume of ice-cold TFB1*.
• Repeat the centrifugationstep. Resuspend the pellet in 1/25 volume of ice-cold TFB2**.
• the cells are aliquoted 100 µL per tube and stored at -80 °C.
* Solution TFB1: 30 mM potassium acetate, 10 mM CaCl2, 50 mM MnCl2, 100 mM RbCl, and 15% glycerol; adjust to pH 5.8 (with 1 M acetic acid), filter-sterilize, and store at 4 °C (ready to use) or at room temperature (cool down before use).
** Solution TFB2: 100 mM MOPS (or PIPES), 75 mM CaCl2, 10 mM RbCl, and 15% glycerol; adjust to pH 6.5 (with 1 M KOH), filter-sterilize, and store at 4 °C (ready to use) or at room temperature (cool down before use).
Transformation of chemical competent E. coli
• Frozen chemically competent cells (100 µL per tube) are thawed on ice.
• The entire ligation (or other DNA sample to be transformed) is added to the cells, and the mix incubated on ice for 30 minutes.
• The cells and DNA mix is heat shocked for 90 s at 42 °C.
•Allow the cells to recover on ice for 5 minutes.
• Add 1 mL of LB medium to the cells and incubate the tube at 37 °C in a shaker-incubator (150 rpm) for 45 min to 1h.
• Plate the appropriate amount of cells on selective LB agar.
Plasmid DNA extraction
