Difference between revisions of "Team:BOKU-Vienna/Protocol"
| Line 237: | Line 237: | ||
<div class="container text-center content-section Protocol"> | <div class="container text-center content-section Protocol"> | ||
<div class="col-lg-8 col-lg-offset-2"> | <div class="col-lg-8 col-lg-offset-2"> | ||
| − | <h2>Methods</h2 | + | <h2>Methods</h2> |
| − | <h4 id="competentcoli" class="test" >Preparation of chemical competent <i>E. coli</i></h4> | + | <h4 id="competentcoli" class="test" >Preparation of chemical competent <i>E. coli</i></h4><br> |
<p> • <i>E. coli </i> 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. <br> • Inoculate 10 mL of LB medium with a single colony and incubate the flask overnight in a shaker-incubator (37°C, shaking 180 rpm). <br> • The following day, transfer 2 mL of this culture to a flask containing 200 mL LB medium and incubate for about 2h until OD<sub>600</sub> raches 0.6. <br> • 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*. <br> • Repeat the centrifugationstep. Resuspend the pellet in 1/25 volume of ice-cold TFB2**. <br> • the cells are aliquoted 100 µL per tube and stored at -80 °C. <br> * Solution TFB1: 30 mM potassium acetate, 10 mM CaCl<sub>2</sub>, 50 mM MnCl<sub>2</sub>, 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). <br> ** Solution TFB2: 100 mM MOPS (or PIPES), 75 mM CaCl<sub>2</sub>, 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).</p> | <p> • <i>E. coli </i> 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. <br> • Inoculate 10 mL of LB medium with a single colony and incubate the flask overnight in a shaker-incubator (37°C, shaking 180 rpm). <br> • The following day, transfer 2 mL of this culture to a flask containing 200 mL LB medium and incubate for about 2h until OD<sub>600</sub> raches 0.6. <br> • 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*. <br> • Repeat the centrifugationstep. Resuspend the pellet in 1/25 volume of ice-cold TFB2**. <br> • the cells are aliquoted 100 µL per tube and stored at -80 °C. <br> * Solution TFB1: 30 mM potassium acetate, 10 mM CaCl<sub>2</sub>, 50 mM MnCl<sub>2</sub>, 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). <br> ** Solution TFB2: 100 mM MOPS (or PIPES), 75 mM CaCl<sub>2</sub>, 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).</p> | ||
Revision as of 13:29, 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).
