Difference between revisions of "Team:IIT Delhi/recipe"

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<h2 id="pfont">Accurate Experimenting for Accurate Results !!</h2>
 
<h2 id="pfont">Accurate Experimenting for Accurate Results !!</h2>
 
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           <button class="accordion back1" style="font-weight: bold;">Transformation</button>
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           <button class="accordion back1" style="font-weight: bold;">Preparation of TAE buffer</button>
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<!--<u><b><h4 id="pfont" style="font-size: 90%;">Preparation of competent  cells:-</h4></b></u>-->
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          <h2 id="pfont"> 20x TAE buffer </h2>
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<b id="pfont">Preparation of competent  cells:-</b>
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          <ul>
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          <li>80mL of 1M Tris solution
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<ol ><left>
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          <li>0.7264g of EDTANa2 salt
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<li>Dilute an overnight culture of E. coli 1:200 with LB broth.
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          <li>2.284 mL glacial acetic acid
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<li>Incubate at 37°C with shaking (at 200 rpm) until the cells reach early log phase (OD600 = 0.25-0.4).
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          <li>distilled H2O 100mL
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<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>
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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)
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<li> Remove the supernatant and discard. Add 0.2 ml of the ice-cold 1X TSS and place the tubes on ice.
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<li>Gently suspend the cells by pipetting.
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<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.
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<b id="pfont">Transformation:-</b>
 
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<br>
 
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<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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           <button class="accordion back2" style="font-weight: bold;">Section 2</button>
 
           <button class="accordion back2" style="font-weight: bold;">Section 2</button>
 
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Revision as of 20:00, 31 October 2017

iGEM IIT Delhi

Description Results Interlab
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Overview Simulation Database
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The Team Attributions

RECIPES

                                                                                                                                                                                                                 

Accurate Experimenting for Accurate Results !!

20x TAE buffer

  • 80mL of 1M Tris solution
  • 0.7264g of EDTANa2 salt
  • 2.284 mL glacial acetic acid
  • distilled H2O 100mL

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