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Team:Lethbridge Canada/notebook april
From 2013hs.igem.org
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</head> | </head> | ||
<body> | <body> | ||
| - | <div id=" | + | <div id="notebook_april"> |
<div id="wrapper"> | <div id="wrapper"> | ||
<div id="header"> | <div id="header"> | ||
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</div> | </div> | ||
| - | <div id=" | + | <div id="cont"> |
<div class="cleafix>"> | <div class="cleafix>"> | ||
<div id="navigation"> | <div id="navigation"> | ||
| - | + | <li class="navigation_project"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/project" class="strict">Project</a> | |
| - | <li class="navigation_project"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/project">Project</a> | + | |
<ul> | <ul> | ||
| - | <li></li> | + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/project">Description</a></li> |
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/project#video_oxy">Visual Modeling</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/math">Math Model</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/results">Results</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/wikifreeze">Wikifreeze</a></li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
| - | <li class="navigation_parts"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/parts">Parts</a> | + | <li class="navigation_parts decrease_opacity"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/parts" class="strict">Parts</a></li> |
| + | <li class="navigation_notebook decrease_opacity"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/protocols" class="strict">Notebook</a> | ||
<ul> | <ul> | ||
| - | <li></li> | + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/protocols#protocol_header">Protocols</a></li> |
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march">Notebook: March</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april">Notebook: April</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may">Notebook: May</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june">Notebook: June</a></li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
| - | <li class=" | + | <li class="navigation_safety decrease_opacity"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/safety" class="strict">Safety</a></li> |
| - | <li class=" | + | <li class="navigation_outreach decrease_opacity"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/outreach" class="strict">Outreach</a> |
| - | + | <ul> | |
| - | <li class="navigation_team"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/team">Team</a></li> | + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/outreach">Overview</a></li> |
| - | <li class="navigation_sponsors"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/sponsors">Sponsors</a></li> | + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/presentations">Presentations</a></li> |
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/interviews">Interviews</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/videos">Videos</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/surveys">Parent Surveys</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/novel_study">Novel Study</a></li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="navigation_team decrease_opacity"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/team" class="strict">Team</a> | ||
| + | <ul> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/team#the_team">The Team</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/team#the_advisors">The Advisors</a></li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="navigation_sponsors decrease_opacity"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/sponsors" class="strict">Sponsors</a></li> | ||
</ul> | </ul> | ||
</div> | </div> | ||
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<div id="notebook_header"> | <div id="notebook_header"> | ||
<div id="notebook_links"> | <div id="notebook_links"> | ||
| - | <h1 id="notebook_links_header">Notebook Links</h1> | + | |
| + | <h1 id="notebook_links_header"> Notebook Links</h1> | ||
| + | |||
<ul id="notebook_march_links"> | <ul id="notebook_march_links"> | ||
| - | <li class="notebook_link_title"><a href=" | + | <li class="notebook_link_title"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march">March</a></li> |
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_one">March 12, 2013: Transformation</a></li> | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_one">March 12, 2013: Transformation</a></li> | ||
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_two">March 13, 2013: Picking Cell Colonies (Cultures)</a></li> | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_two">March 13, 2013: Picking Cell Colonies (Cultures)</a></li> | ||
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<ul id="notebook_april_links"> | <ul id="notebook_april_links"> | ||
| - | <li class="notebook_link_title"><a href=" | + | <li class="notebook_link_title"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april">April</a></li> |
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_one">April 2, 2013: Restriction - Ligation</a></li> | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_one">April 2, 2013: Restriction - Ligation</a></li> | ||
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_two">April 2, 2013: Ligation</a></li> | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_two">April 2, 2013: Ligation</a></li> | ||
| Line 103: | Line 126: | ||
<ul id="notebook_may_links"> | <ul id="notebook_may_links"> | ||
| - | <li class="notebook_link_title"><a href=" | + | <li class="notebook_link_title"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may">May</a></li> |
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_one">May 1, 2013: Ligation</a></li> | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_one">May 1, 2013: Ligation</a></li> | ||
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_two">May 4, 2013: Restriction Digest</a></li> | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_two">May 4, 2013: Restriction Digest</a></li> | ||
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_eighteen">May 17, 2013: Miniprep</a></li> | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_eighteen">May 17, 2013: Miniprep</a></li> | ||
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_nineteen">May 17, 2013: Glycerol Stock</a></li> | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_nineteen">May 17, 2013: Glycerol Stock</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_one">May 22, 2013: Ligation</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_two">May 22, 2013: Transformations</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_three">May 23, 2013: Transformations</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_four">May 23, 2013: Picked Oxytocin and Transformations</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_five">May 24, 2013: Glycerol Stock</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_six">May 24, 2013: Miniprep</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_seven">May 26, 2013: Miniprep</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_eight">May 26, 2013: Restriction Digestion</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_nine">May 26, 2013: Agarose gel of Digestion</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_thirty">May 26, 2013: Digestion Protocol</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_thirty_one">May 31, 2013: Agarose Gel</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_thirty_two">May 31, 2013: Everything</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_thirty_three">May 31, 2013: Everything</a></li> | ||
| + | </ul> | ||
| + | |||
| + | <ul id="notebook_june_links"> | ||
| + | <li class="notebook_link_title"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june">June</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_one">June 1, 2013: Restrictions</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_two">June 2, 2013: Transformation</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_three">June 4, 2013: Picked Colonies</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_four">June 10, 2013: Sent constructs for sequencing</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_five">June 13, 2013: Transformation of OXY into BL21</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_six">June 13, 2013: Restricted and Ligated and Transformed</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_seven">June 13, 2013: Picked Cells</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_eight">June 15, 2013: Ni-Sepharose Purification</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_nine">June 18, 2013: Tris-Tricine PAGE</a></li> | ||
| + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_ten">June 21, 2013: Anti-His Slot Blot</a></li> | ||
</ul> | </ul> | ||
</div> | </div> | ||
</div> | </div> | ||
| - | + | <div class="pull_banner_left"> | |
| + | <img src="https://static.igem.org/mediawiki/2013hs/e/ea/Lethbridge_hs_igem_2013_banner_notbookapril.png"> | ||
| + | </div> | ||
<div id="notebook_body"> | <div id="notebook_body"> | ||
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<li>Names: Yoyo Y, Wesley M</li> | <li>Names: Yoyo Y, Wesley M</li> | ||
<li>Date: April 2, 2013</li> | <li>Date: April 2, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
<li>J06702 - mCherry</li> | <li>J06702 - mCherry</li> | ||
| Line 146: | Line 198: | ||
<li class="notebook_nested_start">Promoter Master Mix</li> | <li class="notebook_nested_start">Promoter Master Mix</li> | ||
<li><ul class="notebook_nested_list"> | <li><ul class="notebook_nested_list"> | ||
| - | <li>MilliQ: 66. | + | <li>MilliQ: 66.25µL</li> |
| - | <li>BSA: 1. | + | <li>BSA: 1.25µL</li> |
| - | <li>Buffer #2: 12. | + | <li>Buffer #2: 12.5µL</li> |
| - | <li>DNA: | + | <li>DNA: 8µL</li> |
| - | <li>Add 0. | + | <li>Add 0.5µL EcoR1 and 0.5µL Spel to 16µL MM</li> |
</ul> | </ul> | ||
</li> | </li> | ||
| Line 156: | Line 208: | ||
<li class="notebook_nested_start">J06702 Master Mix</li> | <li class="notebook_nested_start">J06702 Master Mix</li> | ||
<li><ul class="notebook_nested_list"> | <li><ul class="notebook_nested_list"> | ||
| - | <li>MilliQ: 66. | + | <li>MilliQ: 66.25µL</li> |
| - | <li>BSA: 1. | + | <li>BSA: 1.25µL</li> |
| - | <li>Buffer #2: 12. | + | <li>Buffer #2: 12.5µL</li> |
| - | <li>J06702: | + | <li>J06702: 40µL</li> |
| - | <li>Add 0. | + | <li>Add 0.5µL Xbal and 0.5µL Pst1 to 24MM</li> |
</ul> | </ul> | ||
</li> | </li> | ||
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<li class="notebook_nested_start">pSB1C3 Master Mix</li> | <li class="notebook_nested_start">pSB1C3 Master Mix</li> | ||
<li><ul class="notebook_nested_list"> | <li><ul class="notebook_nested_list"> | ||
| - | <li>MilliQ: 66. | + | <li>MilliQ: 66.25µL</li> |
| - | <li>BSA: 1. | + | <li>BSA: 1.25µL</li> |
| - | <li>Buffer #2: 12. | + | <li>Buffer #2: 12.5µL</li> |
| - | <li>E1010 pSB1C3: | + | <li>E1010 pSB1C3: 40µL</li> |
| - | <li>Add 0. | + | <li>Add 0.5µL EcR1 and 0.5µL Pst1 to 24MM</li> |
</ul> | </ul> | ||
</li> | </li> | ||
</li> | </li> | ||
| - | <li>Incubated at | + | <li>Incubated at 37°C for 1 hour</li> |
| - | <li>Heat killed enzyme for | + | <li>Heat killed enzyme for 20 mins at 65°C</li> |
</ul> | </ul> | ||
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p> | <p class="top_notebook"><a href="#wrapper">Back to Top</a></p> | ||
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<li>Name: Yoyo Y</li> | <li>Name: Yoyo Y</li> | ||
<li>Date: April 2, 2013</li> | <li>Date: April 2, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
<li>Tube 1 - J23100_J06702_pSB1C3</li> | <li>Tube 1 - J23100_J06702_pSB1C3</li> | ||
| Line 195: | Line 247: | ||
<li>Tube 3 - J23113_J06702_pSB1C3</li> | <li>Tube 3 - J23113_J06702_pSB1C3</li> | ||
<li>Tube 4 - J23114_J06702_pSB1C3</li> | <li>Tube 4 - J23114_J06702_pSB1C3</li> | ||
| - | <li>Left overnight at | + | <li>Left overnight at 16°C</li> |
<li>Agarose Gel of Digested Parts 1%</li> | <li>Agarose Gel of Digested Parts 1%</li> | ||
| - | <li><img src=" | + | <li><img src="https://static.igem.org/mediawiki/2013hs/0/05/Lethbridge_hs_igem_2013-4-2-agarose-picture.gif" alt=" " width="400px" /></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 290: | Line 342: | ||
<li>Name: Fiona S, Chris I, Yoyo Y</li> | <li>Name: Fiona S, Chris I, Yoyo Y</li> | ||
<li>Date: April 3, 2013</li> | <li>Date: April 3, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
<li>Antibiotic: CAM</li> | <li>Antibiotic: CAM</li> | ||
| Line 307: | Line 359: | ||
<li>Names: Chris I, Fiona S</li> | <li>Names: Chris I, Fiona S</li> | ||
<li>Date: April 4, 2013</li> | <li>Date: April 4, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
<li>Part Number and Plasmid: J23100_J06702_pSB1C3, J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23114_J06702_pSB1C3</li> | <li>Part Number and Plasmid: J23100_J06702_pSB1C3, J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23114_J06702_pSB1C3</li> | ||
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<li>Names: Chris I, Yoyo Y</li> | <li>Names: Chris I, Yoyo Y</li> | ||
<li>Date: April 5, 2013</li> | <li>Date: April 5, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
| - | <li>Miniprepped J23108_pSB1A2, J23113_pSB1A2, J23114_pSB1A2, J06702_pSB1AK2, used | + | <li>Miniprepped J23108_pSB1A2, J23113_pSB1A2, J23114_pSB1A2, J06702_pSB1AK2, used 50µL of Elution Buffer</li> |
</ul> | </ul> | ||
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p> | <p class="top_notebook"><a href="#wrapper">Back to Top</a></p> | ||
| Line 339: | Line 391: | ||
<li>Names: Chris I, Yoyo Y, Elaine B</li> | <li>Names: Chris I, Yoyo Y, Elaine B</li> | ||
<li>Date: April 5, 2013</li> | <li>Date: April 5, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
</ul> | </ul> | ||
| Line 403: | Line 455: | ||
<li>Names: Shikhar M, Krista F, Austin K</li> | <li>Names: Shikhar M, Krista F, Austin K</li> | ||
<li>Date: April 6, 2013</li> | <li>Date: April 6, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
| - | <li>Miniprepped J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23100_J06702_pSB1C3 used | + | <li>Miniprepped J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23100_J06702_pSB1C3 used 50µL of Elution Buffer</li> |
<li>Note: left plates to grow longer, need DNA sequenced.</li> | <li>Note: left plates to grow longer, need DNA sequenced.</li> | ||
</ul> | </ul> | ||
| Line 420: | Line 472: | ||
<li>Name: Erin K</li> | <li>Name: Erin K</li> | ||
<li>Date: April 7, 2013</li> | <li>Date: April 7, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
| - | <li>J23108_J61002, J23113_J61002, J23114_J61002, | + | <li>J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1AK2</li> |
| - | <li>Separately added | + | <li>Separately added 2µL of appropriate DNA and 0.2µL of Pfu Polymerase to 17.8µL MM</li> |
<li>Agarose Gel confirmation of PCR: Success</li> | <li>Agarose Gel confirmation of PCR: Success</li> | ||
</ul> | </ul> | ||
| Line 435: | Line 487: | ||
<tr> | <tr> | ||
<th>MM 5x Reagent</th> | <th>MM 5x Reagent</th> | ||
| - | <th>Volume( | + | <th>Volume(µL)</th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
| Line 473: | Line 525: | ||
<li>Name: Erin K</li> | <li>Name: Erin K</li> | ||
<li>April 8, 2013</li> | <li>April 8, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
| - | <li>2 samples of J06702_pSB1AK2: Added 0. | + | <li>2 samples of J06702_pSB1AK2: Added 0.5µL XbaI and 0.5µL PstI to 24µL MM</li> |
<li> | <li> | ||
<table class="table_two_left"> | <table class="table_two_left"> | ||
| Line 482: | Line 534: | ||
<tr> | <tr> | ||
<th>J06702 MM 2x</th> | <th>J06702 MM 2x</th> | ||
| - | <th>Volume ( | + | <th>Volume (µL)</th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
| Line 505: | Line 557: | ||
</table> | </table> | ||
</li> | </li> | ||
| - | <li>2 samples of TAT-E1010_pSB1C3: Added 0. | + | <li>2 samples of TAT-E1010_pSB1C3: Added 0.5µL EcoRIand 0.5µL PstI to 24µL MM</li> |
<li> | <li> | ||
<li> | <li> | ||
| Line 512: | Line 564: | ||
<tr> | <tr> | ||
<th>TAT-E1010_pSB1C3 MM 2x</th> | <th>TAT-E1010_pSB1C3 MM 2x</th> | ||
| - | <th>Volume ( | + | <th>Volume (µL)</th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
| Line 536: | Line 588: | ||
</li> | </li> | ||
</li> | </li> | ||
| - | <li>2 samples of each of the 3 promoters: Added | + | <li>2 samples of each of the 3 promoters: Added 5µL appropriate DNA and 0.5µL EcoRI and 0.5µL SpeI to 16µL MM</li> |
<li> | <li> | ||
<table class="table_two_left"> | <table class="table_two_left"> | ||
| Line 542: | Line 594: | ||
<tr> | <tr> | ||
<th>Promoter MM 7x</th> | <th>Promoter MM 7x</th> | ||
| - | <th>Volume ( | + | <th>Volume (µL)</th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
| Line 573: | Line 625: | ||
<li>Names: Wesley M, Riley M, Krista F</li> | <li>Names: Wesley M, Riley M, Krista F</li> | ||
<li>Date: April 8, 2013</li> | <li>Date: April 8, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
| - | <li>Parts: J23108_J61002, J23113_J61002, J23114_J61002, | + | <li>Parts: J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1AK2</li> |
<li>Results: Unsuccessful</li> | <li>Results: Unsuccessful</li> | ||
</ul> | </ul> | ||
| Line 670: | Line 722: | ||
<li>Names: Wesley M, Riley M, Krista F</li> | <li>Names: Wesley M, Riley M, Krista F</li> | ||
<li>Date: April 8, 2013</li> | <li>Date: April 8, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
| - | <li>Protocol: Biobasic Kit: Eluted with | + | <li>Protocol: Biobasic Kit: Eluted with 30µL elution buffer</li> |
</ul> | </ul> | ||
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p> | <p class="top_notebook"><a href="#wrapper">Back to Top</a></p> | ||
| Line 685: | Line 737: | ||
<li>Name: Elaine B, Chris I, Fiona S, Wesley M, Riley M</li> | <li>Name: Elaine B, Chris I, Fiona S, Wesley M, Riley M</li> | ||
<li>Date: April 9, 2013</li> | <li>Date: April 9, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
| - | <li>J23108_J61002, J23113_J61002, J23114_J61002, | + | <li>J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1AK2</li> |
| - | <li>Separately added | + | <li>Separately added 2µL of appropriate DNA and 0.2µL of Pfu Polymerase to 17.8µL MM</li>\ |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 738: | Line 790: | ||
<li>Names: Chris I, Elaine B, Fiona S, Wesley M, Riley M,</li> | <li>Names: Chris I, Elaine B, Fiona S, Wesley M, Riley M,</li> | ||
<li>Date: April 9, 2013</li> | <li>Date: April 9, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
<li>Picked: J23113_J61002, J23108_J61002, J23114_J61002, J23100_J61002, J06702_pSB_1AK2</li> | <li>Picked: J23113_J61002, J23108_J61002, J23114_J61002, J23100_J61002, J06702_pSB_1AK2</li> | ||
| - | <li> | + | <li>5mL cultures, 5µL Amp. grown overnight at 37°C</li> |
</ul> | </ul> | ||
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p> | <p class="top_notebook"><a href="#wrapper">Back to Top</a></p> | ||
| Line 754: | Line 806: | ||
<li>Names: Yoyo Y</li> | <li>Names: Yoyo Y</li> | ||
<li>Date: April 10, 2013</li> | <li>Date: April 10, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
| - | <li>Mini Prep of (x2 of | + | <li>Mini Prep of (x2 of 35µL): J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002</li> |
<li>Mini-prepped with EZ-10 columns</li> | <li>Mini-prepped with EZ-10 columns</li> | ||
| - | <li>Eluted | + | <li>Eluted 35µL</li> |
<li>Purified DNA in HS iGEM Kit #1</li> | <li>Purified DNA in HS iGEM Kit #1</li> | ||
</ul> | </ul> | ||
| Line 773: | Line 825: | ||
<li>Names: Dylan S</li> | <li>Names: Dylan S</li> | ||
<li>Date: April 10, 2013</li> | <li>Date: April 10, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
| - | <li>Add | + | <li>Add 33µL DNA to 17µL MM</li> |
| - | <li> | + | <li>17µL MM to promoter tubes</li> |
| - | <li> | + | <li>33µL DNA to promoter tubes</li> |
<li>Same procedure for J06702_pSB1AK3</li> | <li>Same procedure for J06702_pSB1AK3</li> | ||
| - | <li>Incubated at | + | <li>Incubated at 37°C for 2 hours</li> |
| - | <li>Heat killed at | + | <li>Heat killed at 65°C for 20 minutes</li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 830: | Line 882: | ||
<li>Names: Dylan S, Patrick O</li> | <li>Names: Dylan S, Patrick O</li> | ||
<li>Date: April 11, 2013</li> | <li>Date: April 11, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
| - | <li>Mini prep of ( | + | <li>Mini prep of (35µL EB): J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002</li> |
<li>Mini-prepped with EZ-10 columns</li> | <li>Mini-prepped with EZ-10 columns</li> | ||
| - | <li>Eluted | + | <li>Eluted 35µL</li> |
<li>Purified DNA in HS iGEM Kit #1</li> | <li>Purified DNA in HS iGEM Kit #1</li> | ||
</ul> | </ul> | ||
| Line 849: | Line 901: | ||
<li>Names: Wesley M, Yoyo Y</li> | <li>Names: Wesley M, Yoyo Y</li> | ||
<li>Date: April 12, 2013</li> | <li>Date: April 12, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
<li>Restriction of E1010_pSB1C3 at sites E & P</li> | <li>Restriction of E1010_pSB1C3 at sites E & P</li> | ||
| - | <li> | + | <li>50µL rxn</li> |
| - | <li>Add | + | <li>Add 30µL DNA to 20µL MM</li> |
| - | <li>Added 0. | + | <li>Added 0.5µL EcoR1 & Pst1</li> |
| - | <li>Incubated | + | <li>Incubated 2 hours at 37°C</li> |
| - | <li>Heat killed | + | <li>Heat killed 20 minutes at 65°C</li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 897: | Line 949: | ||
<li>Names: Patrick O, Shikhar M</li> | <li>Names: Patrick O, Shikhar M</li> | ||
<li>Date: April 12, 2013</li> | <li>Date: April 12, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
<li>Reculturization of: J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002</li> | <li>Reculturization of: J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002</li> | ||
| - | <li>PCR: Master mix created with: Annealing temperature: 55. | + | <li>PCR: Master mix created with: Annealing temperature: 55.6°C</li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 970: | Line 1,022: | ||
<li>Names: Amrinder G, Steven T</li> | <li>Names: Amrinder G, Steven T</li> | ||
<li>Date: April 12, 2013</li> | <li>Date: April 12, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
| - | <li>Mini prep of ( | + | <li>Mini prep of (35µL EB): J06702_pSB1AK3, J23100_J61002, J23108_J61002 ,J23113_J61002, J23114_J61002</li> |
<li>Mini-prepped with EZ-10 columns</li> | <li>Mini-prepped with EZ-10 columns</li> | ||
| - | <li>Eluted | + | <li>Eluted 35µL</li> |
<li>Purified DNA in HS iGEM Kit #1</li> | <li>Purified DNA in HS iGEM Kit #1</li> | ||
</ul> | </ul> | ||
| Line 988: | Line 1,040: | ||
<li>Names: Fiona S, Katie T</li> | <li>Names: Fiona S, Katie T</li> | ||
<li>Date: April 14, 2013</li> | <li>Date: April 14, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
| - | <li>Mini prep of ( | + | <li>Mini prep of (35µL EB): J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1AK2</li> |
<li>Mini-prepped with EZ-10 columns</li> | <li>Mini-prepped with EZ-10 columns</li> | ||
| - | <li>Eluted | + | <li>Eluted 35µL</li> |
</ul> | </ul> | ||
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p> | <p class="top_notebook"><a href="#wrapper">Back to Top</a></p> | ||
| Line 1,006: | Line 1,058: | ||
<li>Names: Fiona S, Katie T</li> | <li>Names: Fiona S, Katie T</li> | ||
<li>April 14, 2013</li> | <li>April 14, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
<li>Parts: J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002</li> | <li>Parts: J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002</li> | ||
<li>Gel Extraction</li> | <li>Gel Extraction</li> | ||
| - | <li>Parts: J23108_J61002, | + | <li>Parts: J23108_J61002, J23113_J61002</li> |
| - | <li>Eluted 50 | + | <li>Eluted 50 µL</li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,104: | Line 1,156: | ||
<ul class="notebook_entry_list"> | <ul class="notebook_entry_list"> | ||
<li>Master Mix: Yoyo Yao promoters x9</li> | <li>Master Mix: Yoyo Yao promoters x9</li> | ||
| - | <li>35uL DNA, | + | <li>35uL DNA, 14µL MM, 0.5µL Spe1, 0.5µL Pst1</li> |
<li>Date: April 15, 2013</li> | <li>Date: April 15, 2013</li> | ||
</ul> | </ul> | ||
| Line 1,144: | Line 1,196: | ||
<ul class="notebook_entry_list"> | <ul class="notebook_entry_list"> | ||
<li>Master Mix: FS, KT ½ promoter ½ insulin/glucose x17</li> | <li>Master Mix: FS, KT ½ promoter ½ insulin/glucose x17</li> | ||
| - | <li>(Promoters) | + | <li>(Promoters) 25µL DNA, 24µL MM, 0.5µL Spe1, 0.5µL Pst1</li> |
| - | <li>(Insulin/Glucose) | + | <li>(Insulin/Glucose) 25µL DNA, 24µL MM, 0.5µL EcoR1, 0.5µL Pst1</li> |
<li>Date: April 15, 2013</li> | <li>Date: April 15, 2013</li> | ||
</ul> | </ul> | ||
| Line 1,186: | Line 1,238: | ||
<li>Names: Yoyo Y, Erin K</li> | <li>Names: Yoyo Y, Erin K</li> | ||
<li>April 15, 2013</li> | <li>April 15, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
</ul> | </ul> | ||
| Line 1,471: | Line 1,523: | ||
<li>Name: Dylan S</li> | <li>Name: Dylan S</li> | ||
<li>Date: April 17, 2013</li> | <li>Date: April 17, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
</ul> | </ul> | ||
| Line 1,515: | Line 1,567: | ||
<td>5</td> | <td>5</td> | ||
<td>J23100 mlc J06702</td> | <td>J23100 mlc J06702</td> | ||
| - | <td> | + | <td>pSB3C5</td> |
<td>50</td> | <td>50</td> | ||
</tr> | </tr> | ||
| Line 1,521: | Line 1,573: | ||
<td>6</td> | <td>6</td> | ||
<td>J23100 mlc J06702</td> | <td>J23100 mlc J06702</td> | ||
| - | <td> | + | <td>pSB3C5</td> |
<td>50</td> | <td>50</td> | ||
</tr> | </tr> | ||
| Line 1,545: | Line 1,597: | ||
<li>Name: Chris I</li> | <li>Name: Chris I</li> | ||
<li>Date: April 30, 2013</li> | <li>Date: April 30, 2013</li> | ||
| - | <li>Species: E. | + | <li>Species: E. coli</li> |
| - | <li>Strain: | + | <li>Strain: DH5α</li> |
<li>Protocol: Ute Kothe</li> | <li>Protocol: Ute Kothe</li> | ||
</ul> | </ul> | ||
Latest revision as of 03:49, 22 June 2013
Notebook Links
- March
- March 12, 2013: Transformation
- March 13, 2013: Picking Cell Colonies (Cultures)
- March 13, 2013: Miniprep and Glycerol Stock
- March 15, 2013: Restriction Digestion
- March 16, 2013: Agarose Gel
- March 18, 2013: Agarose Gel
- March 19, 2013: Transformation
- March 20, 2013: Picking Cell Colonies (Cultures)
- March 21, 2013: Miniprep and Glycerol Stock
- March 27, 2013: Transformation
- March 28, 2013: Transformation
- March 28, 2013: Picking Cell Colonies (Cultures)
- March 29, 2013: Picking Cell Colonies (Cultures)
- March 29, 2013: Picking Cells of -80 Glycerol Stock
- April
- April 2, 2013: Restriction - Ligation
- April 2, 2013: Ligation
- April 3, 2013: Transformation
- April 4, 2013: Re-Plating Transformation
- April 5, 2013: Miniprep of ON Cultures
- April 5, 2013: UV Spectroscopy DNA Concentrations
- April 6, 2013: Miniprep From Ligations
- April 7, 2013: PCR
- April 8, 2013: Restriction Digestion of PCR Parts
- April 8, 2013: 3% Agarose Gel of Digested PCR Parts
- April 8, 2013: Gel Extraction
- April 9, 2013: Repeat PCR
- April 9, 2013: Picking Cell Colonies (Cultures)
- April 10, 2013: Miniprep
- April 10, 2013: Restriction
- April 10, 2013: Agarose Gel of PCR 1%
- April 11, 2013: Miniprep
- April 12, 2013: Restriction Digestion
- April 12, 2013: PCR and Cell Reculturization
- April 12, 2013: Miniprep
- April 14, 2013: Miniprep
- April 14, 2013: 1% Agarose Gel
- April 15, 2013: Restriction/Digestion
- April 15, 2013: Restriction/Digestion
- April 15, 2013: 1% Agarose Gel of Promoters
- April 17, 2013: Transformation
- April 30, 2013: 1% Agarose Gel of Promoter Constructs
- May
- May 1, 2013: Ligation
- May 4, 2013: Restriction Digest
- May 4, 2013: 1% Agarose Gel of Promoter Constructs
- May 4, 2013: Gel Extractions
- May 5, 2013: Transformation
- May 7, 2013: Restriction
- May 7, 2013: Gel Extraction
- May 7, 2013: Gel Extracted on Conformation Gel
- May 8, 2013: Ligations
- May 9, 2013: PCR of Ligation Mixture
- May 13, 2013: Restriction of PCR Ligations Gel Conformation
- May 14, 2013: Transformation of Ligations
- May 15, 2013: Ligation
- May 15, 2013: Picking Cells
- May 16, 2013: Picking Cells
- May 16, 2013: 10 Fold Miniprep
- May 17, 2013: Agarose Gel
- May 17, 2013: Miniprep
- May 17, 2013: Glycerol Stock
- May 22, 2013: Ligation
- May 22, 2013: Transformations
- May 23, 2013: Transformations
- May 23, 2013: Picked Oxytocin and Transformations
- May 24, 2013: Glycerol Stock
- May 24, 2013: Miniprep
- May 26, 2013: Miniprep
- May 26, 2013: Restriction Digestion
- May 26, 2013: Agarose gel of Digestion
- May 26, 2013: Digestion Protocol
- May 31, 2013: Agarose Gel
- May 31, 2013: Everything
- May 31, 2013: Everything
- June
- June 1, 2013: Restrictions
- June 2, 2013: Transformation
- June 4, 2013: Picked Colonies
- June 10, 2013: Sent constructs for sequencing
- June 13, 2013: Transformation of OXY into BL21
- June 13, 2013: Restricted and Ligated and Transformed
- June 13, 2013: Picked Cells
- June 15, 2013: Ni-Sepharose Purification
- June 18, 2013: Tris-Tricine PAGE
- June 21, 2013: Anti-His Slot Blot
Restriction - Ligation
- Names: Yoyo Y, Wesley M
- Date: April 2, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- J06702 - mCherry
- J61002 - RFP Reporter
- Promoters: J23113, J23108, J23114, J23100
- Cut Promoters at sites E & S
- Cut mCherry at sites X & P
- Cut Destination Plasmid E1010_pSB1C3 sites E&P
- Promoter Master Mix
- MilliQ: 66.25µL
- BSA: 1.25µL
- Buffer #2: 12.5µL
- DNA: 8µL
- Add 0.5µL EcoR1 and 0.5µL Spel to 16µL MM
- J06702 Master Mix
- MilliQ: 66.25µL
- BSA: 1.25µL
- Buffer #2: 12.5µL
- J06702: 40µL
- Add 0.5µL Xbal and 0.5µL Pst1 to 24MM
- pSB1C3 Master Mix
- MilliQ: 66.25µL
- BSA: 1.25µL
- Buffer #2: 12.5µL
- E1010 pSB1C3: 40µL
- Add 0.5µL EcR1 and 0.5µL Pst1 to 24MM
- Incubated at 37°C for 1 hour
- Heat killed enzyme for 20 mins at 65°C
Ligations
- Name: Yoyo Y
- Date: April 2, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- Tube 1 - J23100_J06702_pSB1C3
- Tube 2 - J23108_J06702_pSB1C3
- Tube 3 - J23113_J06702_pSB1C3
- Tube 4 - J23114_J06702_pSB1C3
- Left overnight at 16°C
- Agarose Gel of Digested Parts 1%
-
Lane # Contents Volume 1 1 kb DNA Ladder 6µL 2 E1010 pSB1c3 #4 15µL 3 E1010 pSB1c3 #3 15µL 4 E1010 pSB1c3 #2 15µL 5 E1010 pSB1c3 #1 15µL 6 J06702 #4 15µL 7 J06702 #3 15µL 8 J06702 #2 15µL 9 J06702 #1 15µL 10 J23100 15µL 11 J23108 15µL 12 J23113 15µL 13 J23114 15µL
Transformation
- Name: Fiona S, Chris I, Yoyo Y
- Date: April 3, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- Antibiotic: CAM
- Part Number and Plasmid: J23100_J06702_pSB1C3, J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23114_J06702_pSB1C3
- Results: TBA
Re-Plating Transformation
- Names: Chris I, Fiona S
- Date: April 4, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- Part Number and Plasmid: J23100_J06702_pSB1C3, J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23114_J06702_pSB1C3
- Antibiotic CAM
Miniprep of ON Cultures
- Names: Chris I, Yoyo Y
- Date: April 5, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- Miniprepped J23108_pSB1A2, J23113_pSB1A2, J23114_pSB1A2, J06702_pSB1AK2, used 50µL of Elution Buffer
UV Spectroscopy DNA Concentrations
- Names: Chris I, Yoyo Y, Elaine B
- Date: April 5, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
-
A260 A280 A260/A280 DNA Concentration
at 50-fold
dilution ug/mLJ23108 0.1017 0.0092 11.05435 254.25 J23113 0.1289 0.0338 3.813609 322.25 J23114 0.135 0.0397 3.400504 337.5 J06702-1 0.13 0.0373 3.485255 325
Miniprep From Ligation
- Names: Shikhar M, Krista F, Austin K
- Date: April 6, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- Miniprepped J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23100_J06702_pSB1C3 used 50µL of Elution Buffer
- Note: left plates to grow longer, need DNA sequenced.
PCR
- Name: Erin K
- Date: April 7, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1AK2
- Separately added 2µL of appropriate DNA and 0.2µL of Pfu Polymerase to 17.8µL MM
- Agarose Gel confirmation of PCR: Success
-
MM 5x Reagent Volume(µL) MilliQ H2O 67 10X Pfu Buffer with MgSO4 20 10mM dNTPs 2 VF2 5 V4 5
Restriction Digestion of PCR Parts
- Name: Erin K
- April 8, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- 2 samples of J06702_pSB1AK2: Added 0.5µL XbaI and 0.5µL PstI to 24µL MM
-
J06702 MM 2x Volume (µL) MIlliQ 29.5 BSA 0.5 NEB 10x Buffer 2 5 J06702_pSB1AK2 10 - 2 samples of TAT-E1010_pSB1C3: Added 0.5µL EcoRIand 0.5µL PstI to 24µL MM
-
TAT-E1010_pSB1C3 MM 2x Volume (µL) MIlliQ 12.5 BSA 0.5 NEB 10x Buffer 2 5 TAT-E1010_pSB1C3 30 - 2 samples of each of the 3 promoters: Added 5µL appropriate DNA and 0.5µL EcoRI and 0.5µL SpeI to 16µL MM
-
Promoter MM 7x Volume (µL) MIlliQ 95.75 BSA 1.75 NEB 10x Buffer 2 17.5
3% Agarose Gel of Digested PCR Parts
- Names: Wesley M, Riley M, Krista F
- Date: April 8, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- Parts: J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1AK2
- Results: Unsuccessful
| Lane # | Contents | Volume(µL) |
|---|---|---|
| 1 | Ladder 100bp | 5 |
| 2 | J23108 | 29 |
| 3 | J23108 | 29 |
| 4 | Blank | N/A |
| 5 | J23113 | 29 |
| 6 | Blank | N/A |
| 7 | Blank | N/A |
| 8 | Blank | N/A |
| 9 | J23113 | 29 |
| 10 | Blank | N/A |
| 11 | J23114 | 29 |
| 12 | Blank | N/A |
| 13 | J23114 | 29 |
Gel Extraction
- Names: Wesley M, Riley M, Krista F
- Date: April 8, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Biobasic Kit: Eluted with 30µL elution buffer
Repeat PCR
- Name: Elaine B, Chris I, Fiona S, Wesley M, Riley M
- Date: April 9, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1AK2
- Separately added 2µL of appropriate DNA and 0.2µL of Pfu Polymerase to 17.8µL MM \
-
MM 5x Reagent Volume (μL) MIlliQ 67 10X Pfu Buffer with MgSO4 2 10mM dNTPs 2 VF2 5 V4 5
Picking Cells
- Names: Chris I, Elaine B, Fiona S, Wesley M, Riley M,
- Date: April 9, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- Picked: J23113_J61002, J23108_J61002, J23114_J61002, J23100_J61002, J06702_pSB_1AK2
- 5mL cultures, 5µL Amp. grown overnight at 37°C
Mini Prep
- Names: Yoyo Y
- Date: April 10, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- Mini Prep of (x2 of 35µL): J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
- Mini-prepped with EZ-10 columns
- Eluted 35µL
- Purified DNA in HS iGEM Kit #1
Restriction
- Names: Dylan S
- Date: April 10, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- Add 33µL DNA to 17µL MM
- 17µL MM to promoter tubes
- 33µL DNA to promoter tubes
- Same procedure for J06702_pSB1AK3
- Incubated at 37°C for 2 hours
- Heat killed at 65°C for 20 minutes
-
Promoter MM 50μL rxn x4 Volume (μL) MIlliQ 44 BSA 20 NEB 10 x Buffer 2 20
Miniprep
- Names: Dylan S, Patrick O
- Date: April 11, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- Mini prep of (35µL EB): J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
- Mini-prepped with EZ-10 columns
- Eluted 35µL
- Purified DNA in HS iGEM Kit #1
Restriction Digestion
- Names: Wesley M, Yoyo Y
- Date: April 12, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- Restriction of E1010_pSB1C3 at sites E & P
- 50µL rxn
- Add 30µL DNA to 20µL MM
- Added 0.5µL EcoR1 & Pst1
- Incubated 2 hours at 37°C
- Heat killed 20 minutes at 65°C
-
MM x 2.5 Volume (μL) MIlliQ 22.5 10x Buffer 2 (NEB) 12.5 BSA 12.5
PCR and Cell Reculturization
- Names: Patrick O, Shikhar M
- Date: April 12, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- Reculturization of: J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
- PCR: Master mix created with: Annealing temperature: 55.6°C
-
MM for Promoters (x5) Conditions for tube "a" MIlliQ 67 μL MIlliQ 13.4 μL 10X Pfu Buffer + MgSO4 20 μL 10X Pfu Buffer + MgSO4 4 μL 10mM dNTPs 2 μL 10mM dNTPs 0.4 μL VF2 5 μL VF2 1 μL VR 5 μL VR 1 μL Pfu polymerase 0.2 μL Pfu polymerase 0.2 μL DNA part 2 μL
Miniprep
- Names: Amrinder G, Steven T
- Date: April 12, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- Mini prep of (35µL EB): J06702_pSB1AK3, J23100_J61002, J23108_J61002 ,J23113_J61002, J23114_J61002
- Mini-prepped with EZ-10 columns
- Eluted 35µL
- Purified DNA in HS iGEM Kit #1
Miniprep
- Names: Fiona S, Katie T
- Date: April 14, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- Mini prep of (35µL EB): J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1AK2
- Mini-prepped with EZ-10 columns
- Eluted 35µL
1% Agarose Gel
- Names: Fiona S, Katie T
- April 14, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
- Parts: J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
- Gel Extraction
- Parts: J23108_J61002, J23113_J61002
- Eluted 50 µL
-
Lane # Contents Volume(µL) 1 1 µL Dye, 5µL DNA kB ladder 6 2 J23100_J61002 25 3 J23100_J61002 25 4 J23100_J61002 ~10 5 J23108_J61002 25 6 J23108_J61002 25 7 J23108_J61002 ~10 8 J23113_J61002 25 9 J23113_J61002 25 10 J23113_J61002 ~10 11 J23114_J61002 25 12 J23114_J61002 25 13 J23114_J61002 ~10
Restriction/Digestion
- Master Mix: Yoyo Yao promoters x9
- 35uL DNA, 14µL MM, 0.5µL Spe1, 0.5µL Pst1
- Date: April 15, 2013
-
Master Mix Volume (μL) MIlliQ 36 BSA 45 10x buffer 2 NEB 45
Restriction/Digestion
- Master Mix: FS, KT ½ promoter ½ insulin/glucose x17
- (Promoters) 25µL DNA, 24µL MM, 0.5µL Spe1, 0.5µL Pst1
- (Insulin/Glucose) 25µL DNA, 24µL MM, 0.5µL EcoR1, 0.5µL Pst1
- Date: April 15, 2013
-
Master Mix Volume (μL) MIlliQ 238 BSA 85 10x buffer 2 NEB 85
1% Agarose Gel of Promoters
- Names: Yoyo Y, Erin K
- April 15, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
-
Lane # Contents Volume (μL) 1 1 KB Ladder 6 2 J23100-1 YYSP 25 3 J23100-1 YYSP 25 4 J23100-1 YYSP <10 5 J23100-2 YYSP 25 6 J23100-2 YYSP 25 7 J23100-2 YYSP <10 8 J23108-1 YYSP 25 9 J23108-1 YYSP 25 10 J23108-1 YYSP <10 11 J23108-2 YYSP 25 12 J23108-2 YYSP ~25 -
Lane # Contents Volume (μL) 1 1 KB Ladder 6 2 J23113-1 YYSP 25 3 J23113-1 YYSP 25 4 J23113-2 YYSP 25 5 J23113-2 YYSP 25 6 J23113-2 YYSP <10 7 J23114-1 YYSP 25 8 J23114-1 YYSP 25 9 J23114-2 YYSP 25 10 J23114-2 YYSP 25 -
Lane # Contents Volume (μL) 1 1 KB Ladder 6 2 J23100-1 YYSP 25 3 J23100-1 YYSP 25 4 J23100-1 YYSP 25 5 J23100-2 YYSP 25 6 J23100-2 YYSP <25 7 J23108-1 YYSP 25 8 J23108-1 YYSP 25 9 J23108-1 YYSP 10 10 J23108-2 YYSP 25 11 J23108-2 YYSP 25 -
Lane # Contents Volume (μL) 1 1 KB Ladder 6 2 J23113-1 YYSP 25 3 J23113-1 YYSP 25 4 J23113-2 YYSP 25 5 J23113-2 YYSP 25 6 J23114-1 YYSP <10 7 J23114-1 YYSP 25 8 J23224-1 YYSP 25 9 J23114-2 YYSP 10 10 J23114-2 YYSP 25
Transformation
- Name: Dylan S
- Date: April 17, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
-
Tube # Part # Plasmid Volume of Competent Cells(µL) 1 J23100+J06702 J61002 100 2 J23108+J06702 J61002 100 3 J23113+J06702 J61002 50 4 J23114+J06702 J61002 50 5 J23100 mlc J06702 pSB3C5 50 6 J23100 mlc J06702 pSB3C5 50 +/- Controls PUC19 Amp resistance 50 each
1% Agarose Gel of Promoter Constructs
- Name: Chris I
- Date: April 30, 2013
- Species: E. coli
- Strain: DH5α
- Protocol: Ute Kothe
-
Lane # Contents Volume(µL) 1 1 KB Ladder 6 2 J23100_J61002 25 3 J23100_J61002 25 4 J23100_J61002 25 5 J23100_J61002 25 6 J23108_J61002 25 7 J23108_J61002 25 8 J23108_J61002 25 9 J23108_J61002 25 10 J23108_J61002 <20 11 J23108_J61002 25 13 1 KB Ladder 25 12 J23100_J61002 <20 1 1 KB Ladder 6 2 J23113_J61002 25 3 J23113_J61002 25 4 J23113_J61002 <20 5 J6702_pSB1AK2 20 6 J6702_pSB1AK2 20 7 J6702_pSB1AK2 20 8 J6702_pSB1AK2 25 9 J6702_pSB1AK2 25 10 J6702_pSB1AK2 20 11 J6702_pSB1AK2 20 12 J6702_pSB1AK2 20 13 None N/A
