Line 18: | Line 18: | ||
<div id="compet" style="display: none;"> | <div id="compet" style="display: none;"> | ||
<h3>Preparation of Bacillus subtilis competent cells</h3> | <h3>Preparation of Bacillus subtilis competent cells</h3> | ||
− | <p | + | <p>Streak out the strain to be made competent on an LB agar plate as a large patch and incubate overnight at 30°C</p> |
− | <p | + | <p>The following morning scrape the cell growth off the plate and use to inoculate fresh, pre-warmed, SpC medium (20 ml) to give an OD600 reading of about 0.5. </p> |
− | <p | + | <p>Incubate the culture at 37°C with vigorous aeration and take periodic OD readings (OD600) to assess cell growth.</p> |
− | <p | + | <p>When the rate of cell growth is seen to depart from exponential (i.e. no significant change in cell density over 20-30 min) inoculate 200 ml of pre-warmed, SpII medium with 2 ml of stationary-phase culture and continue incubation at 37°C with slower aeration </p> |
− | <p | + | <p>After 90 min incubation, pellet the cells by centrifugation (8,000 g, 5min) at room temperature.</p> |
− | <p | + | <p>Carefully decant the supernatant into a sterile container and save.</p> |
− | <p | + | <p>Gently resuspended the cell pellet in 18 ml of the saved supernatant and add 2 ml of sterile glycerol; mix gently </p> |
− | <p | + | <p>Aliquot the competent cell (0.5 ml) in sterile tubes, freeze rapidly in liquid nitrogen or a dry-iced/ethanol bath or ice/isopropanol bath and store -70.</p> |
</div> | </div> | ||
Line 32: | Line 32: | ||
<div id="cellprep" style="display: none;"> | <div id="cellprep" style="display: none;"> | ||
<h3>Cells Preparation :</h3> | <h3>Cells Preparation :</h3> | ||
− | <p | + | <p>Thaw competent cells rapidly by immersing frozen tubes in a 37°C water bath</p> |
− | <p | + | <p>Immediately, add one volume of SpII + EGTA to the Thawed cells; mix gently </p> |
− | <p | + | <p>In a sterile test tube add competent cell (0.2~0.5 ml) to the DNA solution (<0.1 ml) and incubate in a roller drum at 37.</p> |
− | <p | + | <p>Dilute the transformed cells as appropriate in T base containing 0.5% glucose and plate immediately onto selective media.</p> |
<div class="image" id="TransfoPic1"/></div> | <div class="image" id="TransfoPic1"/></div> | ||
<h3>Digestion :</h3> | <h3>Digestion :</h3> | ||
− | <p | + | <p>Select restriction enzymes to digest your plasmid. Determine an appropriate reaction buffer by reading the instructions for your enzyme. </p> |
− | <p | + | <p>In a 1.5mL tube combine the following:</p> |
<ol> | <ol> | ||
<li>-> DNA </li> | <li>-> DNA </li> | ||
Line 48: | Line 48: | ||
<li>-> dH2O up to total volume</li> | <li>-> dH2O up to total volume</li> | ||
</ol> | </ol> | ||
− | <p | + | <p>Mix gently by pipetting. </p> |
− | <p | + | <p>Incubate tube at appropriate temperature (usually 37°C) for 1 hour. </p> |
− | <p | + | <p>Always follow the manufacturer’s instructions. </p> |
− | <p | + | <p>To visualize the results of your digest, conduct gel electrophoresis</p> |
<h3>Vector Preparation :</h3> | <h3>Vector Preparation :</h3> | ||
− | <p | + | <p>Combine the following in a PCR or Eppendorf tube:</p> |
<ol> | <ol> | ||
<li>-> 25ng Vector DNA</li> | <li>-> 25ng Vector DNA</li> | ||
Line 62: | Line 62: | ||
<li>-> 0.5-1μL T4 DNA Ligase</li> | <li>-> 0.5-1μL T4 DNA Ligase</li> | ||
<li>-> H20 to a total of 10μL</li></ol> | <li>-> H20 to a total of 10μL</li></ol> | ||
− | <p | + | <p>Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions).</p> |
</div> | </div> | ||
Revision as of 11:50, 15 October 2016
Competent Cells
Transformation
Digestion :
Select restriction enzymes to digest your plasmid. Determine an appropriate reaction buffer by reading the instructions for your enzyme.
In a 1.5mL tube combine the following:
- -> DNA
- -> Restriction Enzyme(s)
- -> Buffer
- -> dH2O up to total volume
Mix gently by pipetting.
Incubate tube at appropriate temperature (usually 37°C) for 1 hour.
Always follow the manufacturer’s instructions.
To visualize the results of your digest, conduct gel electrophoresis
Vector Preparation :
Combine the following in a PCR or Eppendorf tube:
- -> 25ng Vector DNA
- -> 75ng Insert DNA
- -> Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer)
- -> 0.5-1μL T4 DNA Ligase
- -> H20 to a total of 10μL
Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions).