Team:Sheffield/LabProtocols

From 2014.igem.org

(Difference between revisions)
Line 3: Line 3:
<link href='http://fonts.googleapis.com/css?family=Oswald' rel='stylesheet' type='text/css'>
<link href='http://fonts.googleapis.com/css?family=Oswald' rel='stylesheet' type='text/css'>
<link href='http://fonts.googleapis.com/css?family=Lato:300' rel='stylesheet' type='text/css'>
<link href='http://fonts.googleapis.com/css?family=Lato:300' rel='stylesheet' type='text/css'>
 +
<script src="//code.jquery.com/jquery-1.11.1.js"></script>
<style>
<style>
-
/*.pageContent {
+
#contentSub, #footer-box, #catlinks, #search-controls, #p-logo, .printfooter, .firstHeading,.visualClear {display: none;} /*-- hides default wiki settings --*/
-
position: absolute;
+
 
-
top: 0px;
+
#menubar.left-menu.noprint ul li:last-child {
-
width: 100%;
+
display:none;
-
overflow: scroll;
+
}
-
}*/
+
 
 +
#top-section {
 +
border: 0 none;
 +
height: 14px;
 +
z-index: 100;
 +
/*top: 0;*/
 +
/*position: fixed;*/
 +
width: 975px;
 +
left: 50%;
 +
margin-left: -487px;
 +
}
 +
 
 +
#globalWrapper, #content {
 +
/*position: fixed;*/
 +
/*top: 0;*/
 +
width: 100%;
 +
/*height: 100%;*/
 +
border: 0px;
 +
background-color: transparent;
 +
margin: 0px;
 +
padding: 0px;
 +
}
 +
 
 +
html, body, .wrapper {
 +
width: 100%;  
 +
height: 100%;  
 +
background-color: transparent;
 +
}
 +
 
 +
#bodyContent {
 +
background-color: transparent;
 +
/*overflow-y: scroll;*/
 +
height: 100%;
 +
}
 +
 
 +
#navigationBar, #navigationBar ul {
 +
margin: 0 auto;
 +
padding: 0;
 +
}
-
.headerImage {
+
#navigationBar {
position: fixed;
position: fixed;
top: 0px;
top: 0px;
 +
display: table-row;
width: 100%;
width: 100%;
-
height: 200px;
+
min-width: 1000px;
-
overflow: hidden;
+
height: 60px;
-
z-index: -1;
+
list-style: none;
 +
font-family: 'Oswald', sans-serif;
 +
font-size: 16px;
 +
-webkit-transition: background-color 0.5s linear;
 +
-moz-transition: background-color 0.5s linear;
 +
-o-transition: background-color 0.5s linear;
 +
transition: background-color 0.5s linear;
}
}
-
.headerImage img {
+
.menuText {
-
width: 100%;
+
padding-top: 8px;
}
}
-
.pageTitle {
+
#navigationBar.scrolling {
-
text-align: center;
+
background-color: white;
-
font-family: 'Lato', sans-serif;
+
-
font-size: 60px;
+
-
letter-spacing: -2px;
+
-
color: white;
+
-
font-weight: 300;
+
-
margin-top: 100px;
+
-
margin-bottom: 70px;
+
-
border: none;
+
}
}
-
.gap5px {
+
#navigationBar li {
-
padding-top: 5px;
+
display: table-cell;
 +
height: 100%;
 +
vertical-align: middle;
 +
float: right;
 +
text-align: left;
}
}
-
.pageData {
+
#navigationBar > li:hover > ul {
-
width: 960px;
+
visibility: visible;
-
margin-left: auto;
+
opacity: 1;
-
margin-right: auto;
+
transition-delay:0s;
-
padding: 10px 10px 10px 10px;
+
-
font-family: 'Lato', sans-serif;
+
-
font-size: 16px;
+
}
}
-
h2 {
+
#navigationBar > li > ul {
-
font-family: 'Oswald', sans-serif;
+
visibility: hidden;
-
font-size: 30px;
+
position: absolute;
-
border: none;
+
width: 170px;
 +
margin-top: 8px;
 +
opacity: 0;
 +
-webkit-transition: opacity 0.5s linear;
 +
-moz-transition: opacity 0.5s linear;
 +
-o-transition: opacity 0.5s linear;
 +
transition: opacity 0.5s linear;
}
}
-
h3 {
+
#navigationBar li a {
-
font-weight: bold;
+
display: block;
-
font-size: 18px;
+
padding: 10px 10px;
 +
text-decoration: none;
 +
    white-space: nowrap;
 +
    color: white;
 +
    opacity: 0.9;
}
}
-
</style>
 
-
</head>
 
-
<body>
 
-
<div class="headerImage"><img src="https://static.igem.org/mediawiki/2014/3/34/LabProtocols.jpg"></div>
 
-
<!--<div class="pageContent">-->
 
-
<h1 class="pageTitle">Lab Protocols</h1>
 
-
<div class="gap5px"></div>
 
-
<div class="pageSection1">
+
#navigationBar li span {
-
<div class="pageData">
+
}
-
<h2>Protocol 1: Produce LB Broth</h2>
+
-
<h3>Materials and Equipment</h3>
+
-
LB broth mix, sterile water, weighing scales, weighing boat, conical flask, measuring cylinder.
+
-
<h3>Time</h3>
+
-
Prep: 5 minutes<br/>
+
-
Run: 2 hours
+
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Weigh out 2g of LB broth mix in a weighing boat and add into a 250ml conical flask.</li>
+
-
<li>Measure 100ml sterile water into a measuring cylinder and dispense into the same conical flask.</li>
+
-
<li>Swirl.</li>
+
-
<li>Stopper the flask with cotton wool and foil.</li>
+
-
<li>Label on autoclave tape. Format 'iGEM, Room Number'.</li>
+
-
<li>Move the flask(s) to the autoclave to be sterilised.</li>
+
-
</ol>
+
-
</div>
+
-
</div>
+
-
<div class="pageSection2">
+
#navigationBar li span a {
-
<div class="pageData">
+
padding: 0;
-
<h2>Protocol 2: Produce LB Agar</h2>
+
padding-top: 10px;
-
<h3>Materials and Equipment</h3>
+
text-decoration: none;
-
LB Agar mix, sterile water, weighing scales, weighing boat, conical flask, measuring cylinder.
+
    white-space: nowrap;
-
<h3>Time</h3>
+
    color: white;
-
Prep: 5 minutes<br/>
+
    opacity: 0.9;
-
Run: 2 hours
+
}
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Weigh out 3.5g of LB agar mix in a weighing boat and add into a conical flask.</li>
+
-
<li>Measure 100ml sterile water into a measuring cylinder and dispense into the same conical flask.</li>
+
-
<li>Swirl.</li>
+
-
<li>Stopper the flask with cotton wool and foil.</li>
+
-
<li>Label on autoclave tape. Format 'iGEM, Room Number'.</li>
+
-
<li>Move the flask(s) to the autoclave to be sterilised.</li>
+
-
</ol>
+
-
</div>
+
-
</div>
+
-
<div class="pageSection1">
+
#navigationBar li a.scrolling {
-
<div class="pageData">
+
color: black;
-
<h2>Protocol 3: Make overnight starter cultures</h2>
+
}
-
<h3>Materials and Equipment</h3>
+
-
Stripette, bunsen burner, sterile loop, falcon tubes, LB broth.
+
-
<h3>Time</h3>
+
-
Prep: 10 minutes<br/>
+
-
Run: 16 hours
+
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Use a stripette to take 5ml of LB broth from a 250ml conical flask.</li>
+
-
<li>Dispense into a falcon tube.</li>
+
-
<li>Sterilise a metal loop in a flame.</li>
+
-
<li>Take a culture from an agar plate using the sterile loop and put into one of the falcon tubes.</li>
+
-
<li>Agitate.</li>
+
-
<li>Replicate this using scrapings from a clean agar plate and a fresh tube to use as a positive control.</li>
+
-
<li>Put the tubes into the incubator overnight (37c, 150rpm) to grow up the cultures.</li>
+
-
</ol>
+
-
</div>
+
-
</div>
+
-
<div class="pageSection2">
+
#navigationBar li a:hover {
-
<div class="pageData">
+
color: #FBA917;
-
<h2>Protocol 4: Generate chemically competent E. Coli</h2>
+
}
-
<h3>Materials and Equipment</h3>
+
-
LB broth, starter culture, P1000, P200, pipette tips, incubator, cuvettes, spectrophotometer, Virkon, falcon tubes, ice, weighing scales, centrifuge, CaCl2, 20% glycerol, stripette, eppendorf tubes and -80°C freezer.
+
-
<h3>Time</h3>
+
-
Prep: 40 minutes<br/>
+
-
Run: 5 hours
+
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Grow cells
+
-
<ol>
+
-
<li>Take 1ml starter culture and add to 100ml LB broth.</li>
+
-
<li>Incubate at 37°C, 150rpm.</li>
+
-
<li>Check every hour by testing the optical density at 600nm (OD) using a spectrophotometer to determine whether enough cells are present in the culture.</li>
+
-
<li>0.600 OD is ideal, this is the point at which the cells are in the exponential growth phase.</li>
+
-
<li>Take 1ml of culture into a cuvette to measure; dispose of this after use in Virkon.</li>
+
-
</ol>
+
-
</li>
+
-
<li>Remove cells
+
-
<ol>
+
-
<li>Pour 30ml aliquots from the flasks into falcon tubes; 3 tubes per flask.</li>
+
-
<li>Put tubes on ice for approx 10mins; all equipment used from this point on must be cold e.g. pipette tips.</li>
+
-
<li>Weigh the falcon tubes and pair together similar weight tubes for balance in the centrifuge; tubes paired together must weigh within 0.5g of each other.</li>
+
-
<li>Spin at 4°C, 4000rpm for 5mins.</li>
+
-
<li>After the tubes have all been spun, pour off the supernatant to remove the LB broth, leaving cells in a pellet. Put tubes back on the ice.</li>
+
-
</ol>
+
-
</li>
+
-
<li>Make cells competent
+
-
<ol>
+
-
<li>Add 1ml of CaCl2 to the cells, use the pipette to pull the liquid and cells up and down to resuspend.</li>
+
-
<li>Once resuspended, add another 14ml of CaCl2; 15ml total volume.</li>
+
-
<li>Put back on ice for approx. 10mins to allow cells to acclimatise at the temperature with the CaCl2.</li>
+
-
<li>Re-weigh and pairs tubes again for balance.</li>
+
-
<li>Spin again at 4°C, 4000rpm for 5mins.</li>
+
-
<li>After tubes have been spun, leave on ice for 5mins. Pour off the supernatant?</li>
+
-
</ol>
+
-
</li>
+
-
<li>Aliquot
+
-
<ol>
+
-
<li>Add 600μl of 20% glycerol to each falcon tube.</li>
+
-
<li>Label eppendorf tubes.</li>
+
-
<li>Aliquot 200μl from each falcon tube into eppendorf tubes (3 per falcon).</li>
+
-
<li>Freeze at -80°C.</li>
+
-
</ol>
+
-
</li>
+
-
</ol>
+
-
</div>
+
-
</div>
+
-
<div class="pageSection1">
+
#navigationBar > li > ul > li {
-
<div class="pageData">
+
width: 100%;
-
<h2>Protocol 5: Mini-Prep</h2>
+
margin: 0 0 0 0;
-
<h3>Materials and Equipment</h3>
+
}
-
Ice, starter cultures, mini-prep kit, centrifuge, P100 pipette, weighing scale, Virkon.
+
-
<h3>Time</h3>
+
-
Prep: ?<br/>
+
-
Run: ?
+
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Extract cells
+
-
<ol>
+
-
<li>Match up weights of falcon tubes so they are paired within 0.5g.</li>
+
-
<li>Spin down starter cultures in a centrifuge; 5mins, 4°C, 4000rpm.</li>
+
-
<li>Pour off supernatant into Virkon.</li>
+
-
</ol>
+
-
</li>
+
-
<li>Resuspend cells
+
-
<ol>
+
-
<li>Add 250μl of P1 resuspension buffer to each falcon tube using P1000.</li>
+
-
<li>Resuspend the cells.</li>
+
-
<li>Use pipette to move suspension into separate, labelled eppendorf tubes.</li>
+
-
</ol>
+
-
</li>
+
-
<li>Lyse cells
+
-
<ol>
+
-
<li>Add 250μl of P2 buffer to each eppendorf tube.</li>
+
-
<li>This will lyse cells - a blue colour will indicate they have been lysed.</li>
+
-
<li>Do not leave for more than 5mins.</li>
+
-
</ol>
+
-
</li>
+
-
<li>Neutralise cells
+
-
<ol>
+
-
<li>Add 350μl of N3 neutralisation buffer.</li>
+
-
<li>Once the reaction is complete, the liquid will turn clear/white.</li>
+
-
</ol>
+
-
</li>
+
-
<li>Purify DNA
+
-
<ol>
+
-
<li>Spin down the cells in a centrifuge; 10 mins, 17000g, 4°C.</li>
+
-
<li>Pour supernatant into mini-prep columns.</li>
+
-
<li>Centrifuge columns for 1 min, 17000g, 4°C.</li>
+
-
<li>Discard flow through.</li>
+
-
<li>Add 500μl of PB buffer to each column.</li>
+
-
<li>Centrifuge columns for 1min, 17000g, 4°C.</li>
+
-
<li>Discard flow through.</li>
+
-
<li>Add 750μl PE buffer to each column.</li>
+
-
<li>Centrifuge columns for 1min, 17000g, 4°C.</li>
+
-
<li>Pour off supernatent.</li>
+
-
<li>Centrifuge columns for 1min, 17000g, 4°C.</li>
+
-
<li>Discard bottom of the mini-prep.</li>
+
-
<li>Move column to a new labelled eppendorf.</li>
+
-
<li>Add 50μl of elution buffer.</li>
+
-
<li>Centrifuge columns for 1min, 17000g, 4°C.</li>
+
-
<li>Discard column.</li>
+
-
<li>Immediately place on ice; store in the B56 sewer sample freeze.</li>
+
-
</ol>
+
-
</li>
+
-
</ol>
+
-
</div>
+
-
</div>
+
-
<div class="pageSection2">
+
#navigationBar > li > ul > li > a {
-
<div class="pageData">
+
background-color: #000000;
-
<h2>Protocol 6: Run a gel</h2>
+
opacity: 0.9;
-
<h3>Materials and Equipment</h3>
+
color: white;
-
Agarose mix, TAE buffer, Flask, Microwave, Ethidium bromide, P10, Tips, Autoclave tape, Gel tray, Comb, Buffer tray, Loading buffer, Dna, Eppendorf, Transilluminator.
+
}
-
<h3>Time</h3>
+
-
Prep: 30 mintues<br/>
+
-
Run: 1 hour
+
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Make up a 1% agarose gel
+
-
<ol>
+
-
<li>Weigh out 0.4g agarose.</li>
+
-
<li>Measure 40ml TAE buffer.</li>
+
-
<li>Add both into a 250ml conical flask.</li>
+
-
<li>Swirl.</li>
+
-
<li>Microwave on full for 2 minute, swirling at intervals to ensure all the agarose has dissolved.</li>
+
-
<li>Run side of flask under tap to cool, until comfortable to hold in a gloved hand.</li>
+
-
</ol>
+
-
</li>
+
-
<li>Prepare the gel
+
-
<ol>
+
-
<li>Add 1μl ethidium bromide to the conical flask containing the melted agarose.</li>
+
-
<li>Pour this solution smoothly into a gel tray (sealed with autoclave tape) and add a comb which allows for either 8 or 13 DNA samples to be run at once.</li>
+
-
<li>Leave the gel to set for approximately 15 - 30 minutes.</li>
+
-
<li>Once the gel is set remove the comb and autoclave tape.</li>
+
-
<li>Place the gel tray inside the buffer tray and fill the remaining space with TAE buffer.</li>
+
-
</ol>
+
-
</li>
+
-
<li>Load 'checking' samples
+
-
<ol>
+
-
<li>Load 5μl of 1kn ladder into the first well of the gel.</li>
+
-
<li>Pipette 2μl 5x loading buffer onto a sheet of parafilm.</li>
+
-
<li>pipette 8μl DNA onto the loading buffer and pipette up and down to mix.</li>
+
-
<li>Resting the pipette tip on the back of the well, load 8μl of sample.</li>
+
-
<li>Repeat for each sample.</li>
+
-
<li>Run the Gel at 100v for 60 minutes.</li>
+
-
</ol>
+
-
</li>
+
-
<li>Load 'extraction' samples
+
-
<ol>
+
-
<li>Load 5μl of 1kn ladder into the first well of the gel.</li>
+
-
<li>Add 5x loading buffer into each sample, to give a final ratio of 1:4.</li>
+
-
<li>Resting the pipette tip on the back of the well, load as much sample as possible.</li>
+
-
<li>Repeat for each sample.</li>
+
-
</ol>
+
-
</li>
+
-
<li>Visualise
+
-
<ol>
+
-
<li>Remove the agarose gel from the gel tray and place in the trans-illuminator.</li>
+
-
<li>Open UVP (on the computer desktop).</li>
+
-
<li>Select the type of gel and the exposure time.</li>
+
-
<li>Press capture to take an image.</li>
+
-
<li>Save image to the iGEM2014 folder.</li>
+
-
</ol>
+
-
</li>
+
-
</ol>
+
-
</div>
+
-
</div>
+
-
<div class="pageSection1">
+
#navigationBar > li > ul > li > a.scrolling {
-
<div class="pageData">
+
color: white;
-
<h2>Protocol 7: Pouring Plates</h2>
+
}
-
<h3>Materials and Equipment</h3>
+
-
Sterile hood, Flask, Agar mix, dH2O, antibiotic, empty plates, P200, tips.
+
-
<h3>Time</h3>
+
-
Prep: 15 minutes<br/>
+
-
Run: 20 minutes
+
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Use the sterile hood.</li>
+
-
<li>Make up 100ml LB Agar (Protocol 2).</li>
+
-
<li>Heat in the microwave as follows:
+
-
<ul>
+
-
<li>Power setting 4 for 3 minutes.</li>
+
-
<li>Wait for 6 minutes.</li>
+
-
<li>Power setting 4 for 2 minutes.</li>
+
-
</ul>
+
-
</li>
+
-
<li>Wait to cool (until the flask is warm but comfortable to hold).</li>
+
-
<li>Add 100μl 1/1000 stock of appropriate antibiotic to the agar.</li>
+
-
<li>Pour 4 plates and leave inside the hood to cool and dry.</li>
+
-
</ol>
+
-
</div>
+
-
</div>
+
-
<div class="pageSection2">
+
#igemLogo {
-
<div class="pageData">
+
height: 40px;
-
<h2>Protocol 8: Measure Concentration of DNA with NanoDrop 2000</h2>
+
opacity: 0.8;
-
<h3>Materials and Equipment</h3>
+
padding-right: 10px;
-
Nanodrop, P10, Tips, dH2O, Paper towel.
+
}
-
<h3>Time</h3>
+
-
Prep: 5 minutes<br/>
+
-
Run: 5 minutes
+
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Set up the NanoDrop2000, select “Nucleic Acid”, then “Routine Verification”.</li>
+
-
<li>Spray paper towel with distilled water, then clean metal nodules on metal part and lid.</li>
+
-
<li>Add 1μl of buffer solution to nodule.</li>
+
-
<li>Close lid.</li>
+
-
<li>Select 'Blank' on the programme.</li>
+
-
<li>Reopen lid.</li>
+
-
<li>Add 2μl of DNA Sample.</li>
+
-
<li>Close lid.</li>
+
-
<li>Select 'measure' on the programme.</li>
+
-
<li>Take note of the concentration (in ng/ml).</li>
+
-
<li>Clean nodules again with distilled water.</li>
+
-
<li>Repeat for different DNA sample.</li>
+
-
</ol>
+
-
</div>
+
-
</div>
+
-
<div class="pageSection1">
+
#teamLogo {
-
<div class="pageData">
+
float: left;
-
<h2>Protocol 9: Transforming Cells To Ensure Competency</h2>
+
margin-right: auto;
-
<h3>Materials and Equipment</h3>
+
}
-
Eppendorfs, P1000, P200, P10, Tips, Competent cells, Ligation mix, Ice, Heat block, LB broth, Incubator, Agar plates, Flame, Spreader, Ethanol
+
-
<h3>Time</h3>
+
-
Prep: 5 minutes<br/>
+
-
Run: 1 hour
+
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Using concentration measure by the nanodrop, calculate the volume of DNA sample required: C1V1 = C2V2.</li>
+
-
<li>Where C2 required is 10ng and V2 is 100μl.</li>
+
-
<li>Use the calculated volume of DNA to transform (Protocol 11) chemically competent cells before plating out.</li>
+
-
<li>Calculate the transformation efficiency in colonies per ng of DNA.</li>
+
-
</ol>
+
-
</div>
+
-
</div>
+
-
<div class="pageSection2">
+
#teamLogo li a img {
-
<div class="pageData">
+
height: 80px;
-
<h2>Protocol 10: Blunt End Ligation</h2>
+
-webkit-transition: height 0.25s linear;
-
<h3>Materials and Equipment</h3>
+
-moz-transition: height 0.25s linear;
-
Eppendorfs, P10, tips, Reaction buffer, dH2O, Restricted DNA, T4 Ligase.
+
-o-transition: height 0.25s linear;
-
<h3>Time</h3>
+
transition: height 0.25s linear;
-
Prep: 10 minutes<br/>
+
}
-
Run: 10 minutes
+
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Make up eppendorfs containing ligation mix, as shown:
+
-
<ul>
+
-
<li>10μl reaction buffer.</li>
+
-
<li>8μl water.</li>
+
-
<li>2μl Cut DNA.</li>
+
-
<li>1μl Cut plasmid.</li>
+
-
<li>1μl T4 ligase.</li>
+
-
</ul>
+
-
</li>
+
-
<li>Flick to mix and leave for ten minutes.</li>
+
-
</ol>
+
-
</div>
+
-
</div>
+
-
<div class="pageSection1">
+
#teamLogo li a img.scrolling {
-
<div class="pageData">
+
height: 40px;
-
<h2>Protocol 11: Chemical Tranformation</h2>
+
}
-
<h3>Materials and Equipment</h3>
+
-
Eppendorfs, P1000, P200, P10, Tips, Competent cells, Ligation mix, Ice, Heat block, LB broth, Incubator, Agar plates, Flame, Spreader, Ethanol.
+
-
<h3>Time</h3>
+
-
Prep: 5 minutes<br/>
+
-
Run: 1 hour 45 minutes
+
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Take an appropriate number of eppendorfs, each containing 100μl of competent E. coli cells.</li>
+
-
<li>Into each one, pipette 5μl of each ligation mix.</li>
+
-
<li>Leave a last one to act as a negative control.</li>
+
-
<li>Put the eppendorfs on ice for 30 minutes.</li>
+
-
<li>Heat shock eppendorfs at 42°C for 30 seconds.</li>
+
-
<li>Put on ice for 2 minutes.</li>
+
-
<li>Add 1ml LB broth to each of the eppendorfs and then incubate at 37°C for 60 minutes.</li>
+
-
<li>Remove cells from incubator and spin down at 13000RPM for 60 seconds.</li>
+
-
<li>Pour off the supernatant and resuspend the cells.</li>
+
-
<li>Near a flame, pipette each of the remaining cells onto individual plates (prepared earlier).</li>
+
-
<li>Sterilise a glass spreader using ethanol (flamed) and spread cells evenly until the surface of the agar appears dry.</li>
+
-
<li>Incubate at 37°C overnight.</li>
+
-
</ol>
+
-
</div>
+
-
</div>
+
-
<div class="pageSection2">
+
.pageSection1 {
-
<div class="pageData">
+
background-color: #FFFFFF;
-
<h2>Protocol 12: Sticky End ligation</h2>
+
}
-
<h3>Materials and Equipment</h3>
+
-
Eppendorf, p10, tips, reaction buffer, dH2O, restricted DNA, T4 Ligase.
+
-
<h3>Time</h3>
+
-
Prep: 5 minutes<br/>
+
-
Run: 30 minutes
+
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Make up eppendorfs containing the ligation mix, as follows:
+
-
<ul>
+
-
<li>2μ l reaction buffer.</li>
+
-
<li>10ng insert.</li>
+
-
<li>10ng plasmid.</li>
+
-
<li>1μ l T4 ligase.</li>
+
-
<li>Make up to 20μ l with dH2O.</li>
+
-
</ul>
+
-
</li>
+
-
<li>Flick to mix and leave for 30 minutes.</li>
+
-
</ol>
+
-
</div>
+
-
</div>
+
-
<div class="pageSection1">
+
.pageSection2 {
-
<div class="pageData">
+
background-color: #EBEBEB;
-
<h2>Protocol 13: Restriction Digest</h2>
+
}
-
<h3>Materials and Equipment</h3>
+
-
Eppendorf, p10, p100, tips, restriction enzymes, ice, heat block.
+
-
<h3>Time</h3>
+
-
Prep: 5 minutes<br/>
+
-
Run: 1 hour
+
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>To each eppendorf add:
+
-
<ol>
+
-
<li>10 units of restriction enzyme 1 (usually 1μl).</li>
+
-
<li>10 units of restriction enzyme 2 (usually 1μl).</li>
+
-
<li>1μg DNA.</li>
+
-
<li>5μl 10x buffer.</li>
+
-
<li>Make up to 20μl with dH2O.</li>
+
-
</ol>
+
-
</li>
+
-
<li>Flick to mix.</li>
+
-
<li>Incubate at 37°c for 60 minutes.</li>
+
-
<li>To stop the reaction, heat the sample to an appropriate temperature to inactivate the restriction enzyme used.</li>
+
-
<li>Put on ice.</li>
+
-
</ol>
+
-
</div>
+
-
</div>
+
-
<div class="pageSection2">
+
.socialMediaIcon {
-
<div class="pageData">
+
padding: 0px 0px;
-
<h2>Protocol 14: Glycerol Stocks</h2>
+
margin: 0;
-
<i>Allows stocks of cells to be kept in the -80</i>
+
}
-
<h3>Materials and Equipment</h3>
+
-
P1000 pipette, tips, eppendorf tubes, microwave, 100% glycerol, overnight cultures of cells.
+
-
<h3>Time</h3>
+
-
Prep: 5 minutes<br/>
+
-
Run: 5 minutes
+
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Take 800μl of overnight cells using a P1000 pipette and add into separate, labelled eppendorf tubes.</li>
+
-
<li>Microwave the 100% glycerol for 5 seconds.</li>
+
-
<li>Take 200μl of the glycerol using a P1000 pipette and add into each eppendorf tube.</li>
+
-
<li>Place on ice immediately.</li>
+
-
<li>Transfer to the -80°C freezer to be stored.</li>
+
-
</ol>
+
-
</div>
+
-
</div>
+
-
<!--</div>-->
+
.socialMediaIcon div {
 +
position: relative;
 +
width: 40px;
 +
height: 40px;
 +
}
 +
 
 +
.whiteIcon, .blackIcon, .colouredIcon {
 +
position: absolute;
 +
top: 0;
 +
left: 0;
 +
width: 40px;
 +
height: 40px;
 +
}
 +
 
 +
.whiteIcon {
 +
z-index: 0;
 +
}
 +
 
 +
.blackIcon {
 +
z-index: 1;
 +
visibility: hidden;
 +
}
 +
 
 +
.colouredIcon {
 +
z-index: 2;
 +
visibility: hidden;
 +
}
 +
 
 +
.socialMediaIcon div:hover > .whiteIcon {
 +
visibility: hidden;
 +
}
 +
 
 +
.socialMediaIcon div:hover > .colouredIcon {
 +
visibility: visible;
 +
}
 +
 
 +
.whiteIcon.scrolling {
 +
visibility: hidden;
 +
}
 +
 
 +
.blackIcon.scrolling {
 +
visibility: visible;
 +
}
 +
</style>
 +
<script>
 +
$('#bodyContent').scroll(function() {
 +
console.log("hmm: " + $('#bodyContent').scrollTop());
 +
    if ($('#bodyContent').scrollTop() > 0) {
 +
        $("#navigationBar").addClass("scrolling");
 +
        $("#navigationBar li a").addClass("scrolling");
 +
        $("#teamLogo li a img").addClass("scrolling");
 +
        $("#navigationBar > li > ul > li > a").addClass("scrolling");
 +
        $(".whiteIcon").addClass("scrolling");
 +
        $(".blackIcon").addClass("scrolling");
 +
    } else {
 +
        $("#navigationBar").removeClass("scrolling");
 +
        $("#navigationBar li a").removeClass("scrolling");
 +
        $("#teamLogo li a img").removeClass("scrolling");
 +
        $("#navigationBar > li > ul > li > a").removeClass("scrolling");
 +
        $(".whiteIcon").removeClass("scrolling");
 +
        $(".blackIcon").removeClass("scrolling");
 +
    }
 +
});
 +
</script>
 +
<script>
 +
$(document).ready(function() { // Document ready
 +
console.log("works");
 +
});
 +
</script>
 +
</head>
 +
<body class="content">
 +
<ul id="navigationBar">
 +
<li><a href=""><img id="igemLogo" src="https://static.igem.org/mediawiki/2014/7/7d/IGEMLogo.png"></a></li>
 +
<li>
 +
<span>
 +
<a class="socialMediaIcon" href="http://twitter.com/iGEMSheffield">
 +
<div>
 +
<img class="whiteIcon" src="https://static.igem.org/mediawiki/2014/b/bc/TwitterWhite.png">
 +
<img class="blackIcon" src="https://static.igem.org/mediawiki/2014/b/b2/TwitterBlack.png">
 +
<img class="colouredIcon" src="https://static.igem.org/mediawiki/2014/7/78/TwitterColour.png">
 +
</div>
 +
</a>
 +
</span>
 +
</li>
 +
<li>
 +
<span>
 +
<a class="socialMediaIcon" href="http://www.facebook.com/iGEMSheffield2014">
 +
<div>
 +
<img class="whiteIcon" src="https://static.igem.org/mediawiki/2014/2/29/FacebookWhite.png">
 +
<img class="blackIcon" src="https://static.igem.org/mediawiki/2014/b/b6/FacebookBlack.png">
 +
<img class="colouredIcon" src="https://static.igem.org/mediawiki/2014/1/1b/FacebookColour.png">
 +
</div>
 +
</a>
 +
</span>
 +
</li>
 +
<li class="menuText"><a href="">Attributions &#x25BE;</a>
 +
<ul>
 +
<li><a href="">Acknowledgements</a></li>
 +
<li><a href="">Sponsors</a></li>
 +
</ul>
 +
</li>
 +
<li class="menuText"><a href="">Notebook &#x25BE;</a>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Sheffield/LabProtocols">Protocols</a></li>
 +
<li><a href="">Wet Lab Journal</a></li>
 +
<li><a href="">Dry Lab Journal</a></li>
 +
<li><a href="">Bibliography</a></li>
 +
</ul>
 +
</li>
 +
<li class="menuText"><a href="">Policy and Practices &#x25BE;</a>
 +
<ul>
 +
<li><a href="">Socio Bricks</a></li>
 +
<li><a href="">Lab Notation</a></li>
 +
<li><a href="">Industry Collaboration</a></li>
 +
<li><a href="">SynBio at UoS</a></li>
 +
<li><a href="">Outreach</a></li>
 +
<li><a href="">Collaboration</a></li>
 +
</ul>
 +
</li>
 +
<li class="menuText"><a href="">Results &#x25BE;</a>
 +
<ul>
 +
<li><a href="">FadR Characterisation</a></li>
 +
<li><a href="">Lipase Assay</a></li>
 +
<li><a href="">Keratinase Assay</a></li>
 +
</ul>
 +
</li>
 +
<li class="menuText"><a href="">Modelling &#x25BE;</a>
 +
<ul>
 +
<li><a href="">Product Design</a></li>
 +
<li><a href="">Manufacturing Model</a></li>
 +
<li><a href="">Enzyme Kinetics</a></li>
 +
</ul>
 +
</li>
 +
<li class="menuText"><a href="">Project &#x25BE;</a>
 +
<ul>
 +
<li><a href="">Background</a></li>
 +
<li><a href="">Biosensor</a></li>
 +
<li><a href="">Enzyme Synthesis</a></li>
 +
<li><a href="">Enzyme Secretion</a></li>
 +
<li><a href="">Safety</a></li>
 +
</ul>
 +
</li>
 +
<li class="menuText"><a href="">Team &#x25BE;</a>
 +
<ul>
 +
<li><a href="">Meet the Team</a></li>
 +
<li><a href="">Official Team Profile</a></li>
 +
<li><a href="">Gallery</a></li>
 +
</ul>
 +
</li>
 +
<li class="menuText"><a href="">Home</a></li>
 +
<span id="teamLogo"><li><a href=""><img src="https://static.igem.org/mediawiki/2014/d/da/TeamLogo.png"></a></li></span>
 +
</ul>
</body>
</body>
</html>
</html>
-
 
-
{{Team:Sheffield/NavigationBar}}
 

Revision as of 10:33, 4 October 2014

Retrieved from "http://2014.igem.org/Team:Sheffield/LabProtocols"