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− | <li class="menu_item"> <div class="icon plus"></div> Competent Cells | + | <li class="menu_item"> <div class="icon plus"></div> Competent Cells |
<ul class="submenu"> | <ul class="submenu"> | ||
<li> | <li> | ||
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<h>Preparation of Bacillus subtilis competent cells</h> | <h>Preparation of Bacillus subtilis competent cells</h> | ||
− | <ol> | + | <ol><li>Streak out the strain to be made competent on an LB agar plate as a large patch and incubate overnight at 30°C</li> |
− | < | + | <li>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. </li> |
− | < | + | <li>Incubate the culture at 37°C with vigorous aeration and take periodic OD readings (OD600) to assess cell growth.</li> |
− | < | + | <li>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 </li> |
− | < | + | <li>After 90 min incubation, pellet the cells by centrifugation (8,000 g, 5min) at room temperature.</li> |
− | < | + | <li>Carefully decant the supernatant into a sterile container and save.</li> |
− | < | + | <li>Gently resuspended the cell pellet in 18 ml of the saved supernatant and add 2 ml of sterile glycerol; mix gently </li> |
− | < | + | <li>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.</li></ol> |
</div></a></li></ul></li> | </div></a></li></ul></li> | ||
− | <li class="menu_item"> <div class="icon plus"></div> Transformation | + | <li class="menu_item"> <div class="icon plus"></div> Transformation |
<ul class="submenu"> | <ul class="submenu"> | ||
<li> | <li> | ||
<a href=""> | <a href=""> | ||
<div> | <div> | ||
− | <h | + | <h>Cells Preparation :</h> |
− | <ol> | + | <ol><li>Thaw competent cells rapidly by immersing frozen tubes in a 37°C water bath</li> |
− | < | + | <li>Immediately, add one volume of SpII + EGTA to the Thawed cells; mix gently </li> |
− | < | + | <li>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.</li> |
− | < | + | <li>Dilute the transformed cells as appropriate in T base containing 0.5% glucose and plate immediately onto selective media. </li></ol> |
<p align="center"><img src="https://static.igem.org/mediawiki/igem.org/d/d3/Transfo_pic_1.png" width="400px"/></p> | <p align="center"><img src="https://static.igem.org/mediawiki/igem.org/d/d3/Transfo_pic_1.png" width="400px"/></p> | ||
</div></a> | </div></a> | ||
<a href=""><div> | <a href=""><div> | ||
− | <h>Digestion</h> | + | <h>Digestion :</h> |
− | <ol><li> | + | <ol><li>Select restriction enzymes to digest your plasmid. Determine an appropriate reaction buffer by reading the instructions for your enzyme. </li> |
<p>In a 1.5mL tube combine the following:</p> | <p>In a 1.5mL tube combine the following:</p> | ||
− | <ul><li> | + | <ul><li>DNA </li> |
− | <li> | + | <li>Restriction Enzyme(s)</li> |
− | <li> | + | <li>Buffer </li> |
− | <li> | + | <li>dH2O up to total volume</li> </ul> |
<p>Mix gently by pipetting. </p> | <p>Mix gently by pipetting. </p> | ||
− | <li> | + | <li>Incubate tube at appropriate temperature (usually 37°C) for 1 hour. </li> |
− | <li> | + | <li>Always follow the manufacturer’s instructions. </li> |
− | <li> | + | <li>To visualize the results of your digest, conduct gel electrophoresis</li></ol> |
</div></a> | </div></a> | ||
<a href=""><div> | <a href=""><div> | ||
− | <h>Vector Preparation</h> | + | <h>Vector Preparation :</h> |
<p>Combine the following in a PCR or Eppendorf tube:</p> | <p>Combine the following in a PCR or Eppendorf tube:</p> | ||
− | < | + | <ul><li>25ng Vector DNA</li> |
− | < | + | <li>75ng Insert DNA</li> |
− | < | + | <li>Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer)</li> |
− | < | + | <li>0.5-1μL T4 DNA Ligase</li> |
− | < | + | <li>H20 to a total of 10μL</li></ul> |
<p>Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions).</p> | <p>Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions).</p> | ||
− | </div></a></li></ | + | </div></a></li></ul></li> |
− | <li class="menu_item"> <div class="icon plus"></div> Deletion | + | <li class="menu_item"> <div class="icon plus"></div> Deletion |
<ul class="submenu"> | <ul class="submenu"> | ||
<li> | <li> | ||
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− | <li class="menu_item"> <div class="icon plus"></div> Other | + | <li class="menu_item"> <div class="icon plus"></div> Other |
<ul class="submenu"> | <ul class="submenu"> | ||
<li> | <li> |
Latest revision as of 15:07, 29 July 2016