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

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Interphase by TEMPLATED
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templated.co @templatedco
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Interphase by TEMPLATED
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templated.co @templatedco
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Released for free under the Creative Commons Attribution 3.0 license (templated.co/license)
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table.alt tbody tr td {
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table.alt tbody tr td:first-child {
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table.alt tbody tr:first-child td {
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left: 0;
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#header .container nav {
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right: 0;
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}
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/* Banner */
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#banner {
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background: url("https://static.igem.org/mediawiki/2017/8/8d/T--IIT_DELHI--banner_index.jpg") ;
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background-position: center;
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background-repeat: no-repeat;
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background-size: cover;
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color: #ffffff;
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padding: 41em 0em 1em;
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text-align: center;
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color: #ffffff;
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font-size: 3em;
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line-height: 1em;
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margin: 0 0 0.5em 0;
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padding: 0;
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font-size: 1.5em;
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margin: 3em 0 0 0;
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#banner .button {
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background-color: transparent;
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border: 1px solid #ffffff;
 +
font-weight: 300;
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}
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#banner .button:hover {
 +
background-color: rgba(255, 255, 255, 0.2);
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.wrapper {
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padding: 3em 0em 4em;
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}
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.wrapper.style1 {
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background-color: #3ba666;
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-moz-background-image: linear-gradient(60deg, #4dac71 50%, #3ba666 50%);
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margin-top: 0em;
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.wrapper.style1 header {
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margin-bottom: 4em;
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color: #ffffff;
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border-color: #ffffff;
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margin-bottom: 0.5em;
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}
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.wrapper.style2 .title, .wrapper.style2 header {
 +
border-bottom: medium double rgba(144, 144, 144, 0.25);
 +
padding-bottom: 1em;
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}
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.wrapper.style2 header {
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margin-bottom: 4em;
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}
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.wrapper.style2 .feature {
 +
margin: 0 0 2em 0;
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}
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/* Footer */
 +
 +
#footer {
 +
background: #202222;
 +
color: #ddd;
 +
padding: 1em 0em 1em;
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}
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#footer h2, #footer h3, #footer h4 {
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color: #ffffff;
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#footer a {
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-moz-transition: all 0.2s ease-in-out;
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-webkit-transition: all 0.2s ease-in-out;
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-o-transition: all 0.2s ease-in-out;
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-ms-transition: all 0.2s ease-in-out;
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transition: all 0.2s ease-in-out;
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color: #888;
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#footer a:active, #footer a:hover {
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color: #ffffff;
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font-size: 1.5em;
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border-color: #ffffff;
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#footer .copyright {
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font-size: 0.9em;
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margin: 2em 0 0 0;
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padding: 0;
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text-align: center;
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#footer .copyright li {
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border-left: solid 1px rgba(144, 144, 144, 0.25);
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display: inline-block;
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list-style: none;
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margin-left: 1.5em;
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padding-left: 1.5em;
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border-left: 0;
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margin-left: 0;
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padding-left: 0;
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<style>
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/* ADJUSTS DEFAULT iGEM CONTENT */
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 +
display: none;
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}
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#top_menu_14, #top_menu_inside { /* MAKES BACKGROUND OF iGEM MENU TRANSPARENT */
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background: none;
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border: none;
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#globalWrapper, #content { /* MAKES BACKGROUND OF MEDIAWIKI TRANSPARENT */
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background-color: transparent;
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margin: 0;
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margin-top: -33px;
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padding: 0;
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height: 100%
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html {
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Revision as of 20:31, 28 October 2017

iGEM IIT Delhi

iGEM IIT Delhi
Description Results Proof of Concept Basic Parts Composite Parts
Overview Logic Assessment Signalling Tools
Human Practices Integrated Practices Public Engagement Collaborations
Lab Records Protocols Recipes Medal Criteria Recipes
The Team Attributions



LAB PROTOCOLS

Accurate Experimenting for Accurate Results !!

Preparation of competent cells:-

  1. Dilute an overnight culture of E. coli 1:200 with LB broth.
  2. Incubate at 37°C with shaking (at 200 rpm) until the cells reach early log phase (OD600 = 0.25-0.4).
  3. 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.
    OR
    (if the above 1X TSS is not available)
    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.
  4. 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)
  5. Remove the supernatant and discard. Add 0.2 ml of the ice-cold 1X TSS and place the tubes on ice.
  6. Gently suspend the cells by pipetting.
  7. 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.

Transformation:-


  1. Thaw frozen TSS-competent cells slowly on ice(if stored at -70°C).
  2. Add 100 pg -200 ng (2.5 to 4 ul)(15ul for ligation product)of DNA to each tube of competent cells.
    Note: Addition of more than 10ng of DNA may significantly decrease transformation efficiencies.
  3. Flick the tubes to mix the cells and DNA and incubate the cells on ice for 30 minutes.
  4. Transfer the tubes to water bath/dry bath(42°C) for 90 seconds.
  5. Transfer the tubes to ice and incubate for an additional 10 minutes.
  6. 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).
  7. Centrifuge the cells at 3000g for ~ 6min (10 mins after ligation)at 4deg(in temperature control centrifuge).
  8. Aspirate the tubes to leave the pellets with 1/4 broth .(keep ~300ul)
  9. Plate the cells on-to the appropriate selective or differential medium and incubate overnight at 37°C.Check the procedure for antibiotic.
    1. For Ampicillin: 12ul Amp + 188 ul MQ. In MCT spread it on the culture plate before adding the DNA.
    2. For Chloramphenicol: 1:1000 volume ratio of antibiotic : culture broth. Directly suspend into the culture broth and spread it on the plate.
    3. For Kanamycin: 1:1000 volume ratio of antibiotic : culture broth. Directly suspend into the culture broth and spread it on the plate.
      DNA should be added as soon as the last trace of ice in the tube disappears.
        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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Contact Us Address

E-mail: iitd.igem@gmail.com
Undergraduate Laboratory
Department of Biotechnology and Biochemical Engineering, IIT Delhi