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

Revision as of 20:05, 29 October 2017

iGEM IIT Delhi

LAB PROTOCOLS

Accurate Experimenting for Accurate Results !!

Preparation of competent cells:-

Transformation:-

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 <b id="pfont">Preparation of competent  cells:-</b>
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-<li>Dilute an overnight culture of E. coli 1:200 with LB broth.
-<li>Incubate at 37°C with shaking (at 200 rpm) until the cells reach early log phase (OD600 = 0.25-0.4).
-<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>
-OR <br>
-(if the above 1X TSS is not available)<br>
-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.
-<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)
-<li> Remove the supernatant and discard. Add 0.2 ml of the ice-cold 1X TSS and place the tubes on ice.
-<li>Gently suspend the cells by pipetting.
-<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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-<li>Thaw frozen TSS-competent cells slowly on ice(if stored at -70°C).
-<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.
-<li>Flick the tubes to mix the cells and DNA and incubate the cells on ice for 30 minutes. <br>
-<li>  Transfer the tubes to water bath/dry bath(42°C) for 90 seconds.
-<li> Transfer the tubes to ice and incubate for an additional 10 minutes.
-<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).
-<li> Centrifuge the cells at 3000g for  ~ 6min (10 mins after ligation)at 4deg(in temperature control centrifuge).
-  <li> Aspirate the tubes to leave the pellets with 1/4 broth .(keep ~300ul)
-    <li>Plate the cells on-to the appropriate selective or differential medium and incubate overnight at 37°C.Check the procedure for antibiotic.
-      <ol> <li>For Ampicillin: 12ul Amp + 188 ul MQ. In MCT spread it on the culture plate before adding the DNA.
-        <li>For Chloramphenicol: 1:1000 volume ratio of antibiotic : culture broth. Directly suspend into the culture broth and spread it on the plate.
-          <li> For Kanamycin: 1:1000 volume ratio of antibiotic : culture broth. Directly suspend into the culture broth and spread it on the plate.
-          </ol>
-          <ul> DNA should be added as soon as the last trace of ice in the tube disappears.
-<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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	E-mail: iitd.igem@gmail.com Undergraduate Laboratory Department of Biotechnology and Biochemical Engineering, IIT Delhi