Protocols

From 2008.igem.org

(Difference between revisions)
(New page: A. PCR (25ul) or Wobble PCR (50ul) ::I. PCR (25ul) ::::1. Mastermix: 20.375 ul water, 0.5ul 10mM dNTP, 2.5ul 10x Buffer 2, 0.375ul Expand Polymerase 1. ::::::a. Aliquot 23.75 ul. ::::2. ...)
 
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A. PCR (25ul) or Wobble PCR (50ul)
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<html>
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::I. PCR (25ul)
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<head>
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::::1.  Mastermix: 20.375 ul water, 0.5ul 10mM dNTP, 2.5ul 10x Buffer 2, 0.375ul Expand Polymerase 1.
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<style>
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::::::a. Aliquot 23.75 ul.
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#globalWrapper {
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::::2.  Add 0.5ul Oligo 1 (10uM), 0.5ul Oligo 2 (10uM), 0.25 ul template DNA.
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margin: 0px;
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::::3.  Load into thermocycler.  **Small Holes are for 25ul and Big Holes for 50ul to maximize contact**
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padding: 0px;
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::::4.  Select the program
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background-color: #717144 !important;
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}
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#header {
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background-color: #717144;
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width: 100%;
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height: 180px;
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background: url(https://static.igem.org/mediawiki/2008/7/77/JHU_0708_BubblesLogo.gif) no-repeat 0px top;
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}
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#footer-box {
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border-top-color: #717144;
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border-right-color: #717144;
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border-bottom-color: #717144;
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border-left-color: #717144;
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background: #717144;
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}
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#content{
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background:#717144;
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border-left-color: #717144;
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border-right-color: #717144;
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}
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#leftcol {
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background: #717144;
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float: left;
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width: 160px;
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height: 476px;
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}
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/*#name {
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                        border: 2px solid black;
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                        margin: 20 20 500 0;
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                        height: 30px;
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                        width: 90px;
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                }*/
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div#header a, div#leftcol a {
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position: relative;
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height: 118px;
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width: 118px;
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float: right;
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text-decoration: none;
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}
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        #divholder{
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                        margin-right: 0px;
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                }
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div#header a {
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bottom: 0px;
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margin: 65px 0 0 0;
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}
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                /*div#header a#Biobrick{margin-right:20px;}*/
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::II. Wobble PCR
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/* leftcol nav */
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::::1. Mastermix: 40ul water, 1.5 ul MgCl2, 5ul buffer (Taq), 1ul dNTP 10mM dNTP, 0.5 ul Taq Polymerase
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#Home { background-image: url(https://static.igem.org/mediawiki/2008/b/bd/JHU_0708_HomeB.gif); }
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::::::a. Aliquot 48 ul
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#Home:hover { background-image: url(https://static.igem.org/mediawiki/2008/0/06/JHU_0708_HomeH.gif); }
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::::2. Add 1 ul 100 uM Oligo 1, 1 ul 100 uM Oligo 2.
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#People { background-image: url(https://static.igem.org/mediawiki/2008/1/1b/JHU_0708_PeopleB.gif); }
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::::3. Load and select wobble program
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#People:hover { background-image: url(https://static.igem.org/mediawiki/2008/8/87/JHU_0708_PeopleH.gif); }
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#Money { background-image: url(https://static.igem.org/mediawiki/2008/6/6b/JHU_0708_MoneyB.gif); }
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#Money:hover { background-image: url(https://static.igem.org/mediawiki/2008/1/16/JHU_0708_MoneyH.gif); }
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/* header nav */
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#Research { background-image: url(https://static.igem.org/mediawiki/2008/0/04/JHU_0708_ResearchB.gif); }
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#Research:hover { background-image: url(https://static.igem.org/mediawiki/2008/2/20/JHU_0708_ResearchH.gif); }
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#Notebook { background: url(https://static.igem.org/mediawiki/2008/4/47/JHU_0708_NotebookB.gif); }
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#Notebook:hover { background: url(https://static.igem.org/mediawiki/2008/1/11/JHU_0708_NotebookH.gif) }
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#Biobrick { background: url(https://static.igem.org/mediawiki/2008/0/05/JHU_0708_BiobrickB.gif);}
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#Biobrick:hover { background: url(https://static.igem.org/mediawiki/2008/8/8c/JHU_0708_BiobrickH.gif); }
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#Protocols { background: url(https://static.igem.org/mediawiki/2008/e/ee/JHU_0708_ProtocolsB.gif); }
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#Protocols:hover { background: url(https://static.igem.org/mediawiki/2008/a/a3/JHU_0708_ProtocolH.gif); }
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B. Use Clonewells to isolate DNA samples.  Because sample B5 (K112312) may have been contaminated, PCR for b5 was done again.
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#book {
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float: top;
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background: #FFFF99;
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width: 700px;
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/*height: 85%;*/
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border: 10px solid #FFFF99;
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margin-left: 160px;
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padding-right: 40px;
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padding-left: 10px;
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min-height: 450px;
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}
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}
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font.ir {
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font-size:85%;
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line-height:1.3;
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}
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a{color:#BFBFBF;text-decoration:none;}
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a:hover{color:#5888AB}
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a img{border:0px}
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a:visited {
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color: #000000;
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}
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</style>
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</head>
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C.  Perform digestion using the follow procedure.
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<body>
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<div id="header">
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          <div id="divholder">
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                <div id="name"><font class=ir></font></div>
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<a href="Biobrick" id="Biobrick"></a>
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<a href="Protocols" id="Protocols"></a>
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                <a href="Research" id="Research"></a>
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                <a href="Notebook" id="Notebook"></a>
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          </div>
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</div>
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::1. Make a solution of 5 ul NEB2, 15 ul DNA, 1 ul EcoRI, 1 ul BamHI, 28 ul water
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<div id="leftcol">
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::2. Incubate for an hour
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<a href="https://2008.igem.org/Team:Johns_Hopkins" id="Home"></a>
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<a href="People" id="People"></a>
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<a href="Donations" id="Money"></a>
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</div>
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D.  Clean up digestion with zymo columns.
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<div id="book"><h2>Welcome to Johns Hopkins iGEM</h2>
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::1.  Add 200ul ADB buffer to each of the digestion samples and pipette into zymo columns.  Spin at 15s at full speed.
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<p>
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::2.  Add 200ul wash buffer.  Spin for 15s at full speed.
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<font class=ir>
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::3.  Repeat step 2.  Spin for an additional 90s at full speed.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The International Genetically Engineered Machines Competition (iGEM) is an annual intercollegiate challenge that seeks to answer the question: "Can simple biological systems be built from standard, interchangeable parts and operated in living cells? Or is biology simply too complicated to be engineered in this way?" The newly-formed iGEM team at Johns Hopkins is composed of mostly undergraduate students with diverse majors ranging from Materials Science Engineering to Biology. While the team has graduate and faculty input, it is almost totally undergraduate run. The iGEM teams work over the summer at their, respective university, and then attend the iGEM Jamboree in November to present their work. We plan to create novel genetic parts that could be added to the existing iGEM registry of biological parts. The MIT iGEM Registry of Parts is the databank of biological standards to which iGEM teams from 2003 and onward have contributed. Currently, the databank only has 16 yeast "biobricks". These biobricks are reporters, tags, plasmids and other useful interchangeable genetic parts, that could one day revolutionize genetic and synthetic biology. By the end of our project, we will have most likely doubled the amount of yeast biobricks in the registry.
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::4.  Add 7ul of water to tube and spin for 30s at full speed.
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</font>
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</p>
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E.  Ligation
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<br></br>
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::1.  Mastermix: 6.5ul water, 1ul ligation buffer, 0.5 T4 DNA ligase, 1ul pBca1256.  Aliquot 9ul.
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</div>
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::2.  Add 1ul of insert.
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</body>
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::3.  Cover with foil and incubate for 30 min at room temperature.
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</html>
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F.  Transformation (always keep on ice)
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::1.  220ul competent cell in one tube. Thaw on ice.
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::2.  Add 30ul KCM and 20 ul water (both cold) to each tube of competent cells.
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::3.  Invert ~2x to mix.
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::4. Aliquot 45ul cell into 10 ul of ligation rxn. Swirl and pipette up and down once to mix.
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::5.  Foil all tubes and incubate 10 min in ice-water.
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::6.  Heat shock 90 sec at 42 C.
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::7.  Incubate on ice for 2 min.
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::8.  Rub ethanol and flame top of foil to sterilize. Add 50 ul of LB to each tube by poking holes into each tube with pipet tips.
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::9.  Re-cover all tubes with foil.
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::9.  Incubate for 1 hr at 37 C.
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::10. Plate
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Latest revision as of 04:05, 11 July 2008

Welcome to Johns Hopkins iGEM

        The International Genetically Engineered Machines Competition (iGEM) is an annual intercollegiate challenge that seeks to answer the question: "Can simple biological systems be built from standard, interchangeable parts and operated in living cells? Or is biology simply too complicated to be engineered in this way?" The newly-formed iGEM team at Johns Hopkins is composed of mostly undergraduate students with diverse majors ranging from Materials Science Engineering to Biology. While the team has graduate and faculty input, it is almost totally undergraduate run. The iGEM teams work over the summer at their, respective university, and then attend the iGEM Jamboree in November to present their work. We plan to create novel genetic parts that could be added to the existing iGEM registry of biological parts. The MIT iGEM Registry of Parts is the databank of biological standards to which iGEM teams from 2003 and onward have contributed. Currently, the databank only has 16 yeast "biobricks". These biobricks are reporters, tags, plasmids and other useful interchangeable genetic parts, that could one day revolutionize genetic and synthetic biology. By the end of our project, we will have most likely doubled the amount of yeast biobricks in the registry.



Retrieved from "http://2008.igem.org/Protocols"