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| − | <h2 class="h2font">LAB PROTOCOLS</h2>
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| − | <p> | + | <h2>march</h2> |
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| + | <h2>april</h2> |
| − | <h2 id="pfont">Accurate Experimenting for Accurate Results !!</h2> | + | <p>Lorem ipsum..</p> |
| − | </header>
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| − | <button class="accordion back1" style="font-weight: bold;">Transformation</button>
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| − | <!--<u><b><h4 id="pfont" style="font-size: 90%;">Preparation of competent cells:-</h4></b></u>--> | + | |
| − | <b id="pfont">Preparation of competent cells:-</b> | + | |
| − | <div align="left" style="font-size: 84%;"> | + | <div class=" container1 left1"> |
| − | <ol ><left> | + | <div class="content1"> |
| − | <li>Dilute an overnight culture of E. coli 1:200 with LB broth. | + | <h2>may</h2> |
| − | <li>Incubate at 37°C with shaking (at 200 rpm) until the cells reach early log phase (OD600 = 0.25-0.4). | + | <p>Lorem ipsum..</p> |
| − | <li>We already have 1X TSS in 4 deg fridge(old). Use it without dilution or thawing. Keep it inside the icebox just after taking out from the fridge. <br> | + | </div> |
| − | OR <br>
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| − | (if the above 1X TSS is not available)<br>
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| − | While cells are growing, thaw 2X TSS on ice and dilute an appropriate amount 1:1 with sterile distilled water (100µl of diluted TSS will be needed for each ml of cells). Chill on ice.
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| − | <li> Place 2-ml aliquots of early log-phase cells into sterile 2-ml micro-centrifuge tubes and pellet the cells by centrifugation at 4°C at 3000g for 10 min.(6-8 mins for taking part from Igem kit) | + | <h2>june</h2> |
| − | <li> Remove the supernatant and discard. Add 0.2 ml of the ice-cold 1X TSS and place the tubes on ice. | + | <p>Lorem ipsum..</p> |
| − | <li>Gently suspend the cells by pipetting. | + | </div> |
| − | <li> Proceed with the transformation protocol below (Step 2), or immediately freeze cells by immersion in liquid nitrogen or a dry ice/ethanol bath. Store the frozen cells at –70°C. | + | </div><div class="container1 left1"> |
| − | </left> | + | <div class="content1"> |
| − | </ol> | + | <h2>july</h2> |
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| | + | <h2>august</h2> |
| | + | <p>Lorem ipsum..</p> |
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| | + | <h2>september</h2> |
| | + | <p>Lorem ipsum..</p> |
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| | + | <h2>october</h2> |
| | + | <p>Lorem ipsum..</p> |
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| − | <b id="pfont">Transformation:-</b>
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| − | <br>
| + | </section> |
| − | <div align="left" style="font-size: 84%;">
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| − | <ol ><left> | + | |
| − | <li>Thaw frozen TSS-competent cells slowly on ice(if stored at -70°C).
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| − | <li>Add 100 pg -200 ng (2.5 to 4 ul)(15ul for ligation product)of DNA to each tube of competent cells. <br>Note:Addition of more than 10ng of DNA may significantly decrease transformation efficiencies.
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| − | <li>Flick the tubes to mix the cells and DNA and incubate the cells on ice for 30 minutes. <br>
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| − | <li> Transfer the tubes to water bath/dry bath(42°C) for 90 seconds.
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| − | <li> Transfer the tubes to ice and incubate for an additional 10 minutes.
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| − | <li>Add 800 ul (total 1 mL)of LB broth and incubate the cells at 37°C for up to 1 hour with shaking (at 200 rpm).
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| − | <li> Centrifuge the cells at 3000g for ~ 6min (10 mins after ligation)at 4deg(in temperature control centrifuge).
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| − | <li> Aspirate the tubes to leave the pellets with 1/4 broth .(keep ~300ul)
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| − | <li>Plate the cells on-to the appropriate selective or differential medium and incubate overnight at 37°C.Check the procedure for antibiotic.
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| − | <ol> <li>For Ampicillin: 12ul Amp + 188 ul MQ. In MCT spread it on the culture plate before adding the DNA.
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| − | <li>For Chloramphenicol: 1:1000 volume ratio of antibiotic : culture broth. Directly suspend into the culture broth and spread it on the plate.
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| − | <li> For Kanamycin: 1:1000 volume ratio of antibiotic : culture broth. Directly suspend into the culture broth and spread it on the plate.
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| − | </ol>
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| − | <ul> DNA should be added as soon as the last trace of ice in the tube disappears.
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| − | <ul>Incubate on ice for 30 minutes. Expect a 2-fold loss in TE for every 10 minutes you shorten this step.
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| − | </left>
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