Team:Lethbridge Canada/notebook april

From 2013hs.igem.org

(Difference between revisions)
Line 176: Line 176:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
<li>Protocol: Ute Kothe</li>
<li>Protocol: Ute Kothe</li>
<li>J06702 - mCherry</li>
<li>J06702 - mCherry</li>
Line 187: Line 187:
<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: 66.25uL</li>
+
<li>MilliQ: 66.25µL</li>
-
<li>BSA: 1.25 uL</li>
+
<li>BSA: 1.25µL</li>
-
<li>Buffer #2: 12.5 uL</li>
+
<li>Buffer #2: 12.5µL</li>
-
<li>DNA: 8 uL</li>
+
<li>DNA: 8µL</li>
-
<li>Add 0.5 uL EcoR1 and 0.5 uL Spel to 16 uL MM</li>
+
<li>Add 0.5µL EcoR1 and 0.5µL Spel to 16µL MM</li>
</ul>
</ul>
</li>
</li>
Line 197: Line 197:
<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: 66.25 uL</li>
+
<li>MilliQ: 66.25µL</li>
-
<li>BSA: 1.25 uL</li>
+
<li>BSA: 1.25µL</li>
-
<li>Buffer #2: 12.5 uL</li>
+
<li>Buffer #2: 12.5µL</li>
-
<li>J06702: 40 uL</li>
+
<li>J06702: 40µL</li>
-
<li>Add 0.5 uL Xbal and 0.5 uL Pst1 to 24MM</li>
+
<li>Add 0.5µL Xbal and 0.5µL Pst1 to 24MM</li>
</ul>
</ul>
</li>
</li>
Line 207: Line 207:
<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: 66.25uL</li>
+
<li>MilliQ: 66.25µL</li>
-
<li>BSA: 1.25 uL</li>
+
<li>BSA: 1.25µL</li>
-
<li>Buffer #2: 12.5 uL</li>
+
<li>Buffer #2: 12.5µL</li>
-
<li>E1010 pSB1C3: 40 uL</li>
+
<li>E1010 pSB1C3: 40µL</li>
-
<li>Add 0.5 uL EcR1 and 0.5 uL Pst1 to 24MM</li>
+
<li>Add 0.5µL EcR1 and 0.5µL Pst1 to 24MM</li>
</ul>
</ul>
</li>
</li>
</li>
</li>
-
<li>Incubated at 37 degrees Celsuis for 1 hour</li>
+
<li>Incubated at 37°C for 1 hour</li>
-
<li>Heat killed enzyme for 20mins at 65 degrees Celsius</li>
+
<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>
Line 229: Line 229:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<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 236: Line 236:
<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 16 degrees Celsuis</li>
+
<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="https://static.igem.org/mediawiki/2013hs/0/05/Lethbridge_hs_igem_2013-4-2-agarose-picture.gif" alt=" " width="400px" /></li>
<li><img src="https://static.igem.org/mediawiki/2013hs/0/05/Lethbridge_hs_igem_2013-4-2-agarose-picture.gif" alt=" " width="400px" /></li>
Line 331: Line 331:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
<li>Protocol: Ute Kothe</li>
<li>Protocol: Ute Kothe</li>
<li>Antibiotic: CAM</li>
<li>Antibiotic: CAM</li>
Line 348: Line 348:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<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>
Line 364: Line 364:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<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 50uL of Elution Buffer</li>
+
<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 380: Line 380:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
<li>Protocol: Ute Kothe</li>
<li>Protocol: Ute Kothe</li>
</ul>
</ul>
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<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<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 50uL of Elution Buffer</li>
+
<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 461: Line 461:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
<li>Protocol: Ute Kothe</li>
<li>Protocol: Ute Kothe</li>
<li>J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSb1Ak2</li>
<li>J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSb1Ak2</li>
-
<li>Separately added 2uL of appropriate DNA  and 0.2uL of Pfu Polymerase to 17.8 uL MM</li>
+
<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 476: Line 476:
<tr>
<tr>
<th>MM 5x Reagent</th>
<th>MM 5x Reagent</th>
-
<th>Volume(uL)</th>
+
<th>Volume(µL)</th>
</tr>
</tr>
</thead>
</thead>
Line 514: Line 514:
<li>Name: Erin K</li>
<li>Name: Erin K</li>
<li>April 8, 2013</li>
<li>April 8, 2013</li>
-
<li>Species: E. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
<li>Protocol: Ute Kothe</li>
<li>Protocol: Ute Kothe</li>
-
<li>2 samples of J06702_pSB1AK2: Added 0.5uL XbaI and 0.5uL PstI to 24uL MM</li>
+
<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 523: Line 523:
<tr>
<tr>
<th>J06702 MM 2x</th>
<th>J06702 MM 2x</th>
-
<th>Volume (uL)</th>
+
<th>Volume (µL)</th>
</tr>
</tr>
</thead>
</thead>
Line 546: Line 546:
</table>
</table>
</li>
</li>
-
<li>2 samples of TAT-E1010_pSB1C3: Added 0.5uL EcoRIand 0.5uL PstI to 24uL MM</li>
+
<li>2 samples of TAT-E1010_pSB1C3: Added 0.5µL EcoRIand 0.5µL PstI to 24µL MM</li>
<li>
<li>
<li>
<li>
Line 553: Line 553:
<tr>
<tr>
<th>TAT-E1010_pSB1C3 MM 2x</th>
<th>TAT-E1010_pSB1C3 MM 2x</th>
-
<th>Volume (uL)</th>
+
<th>Volume (µL)</th>
</tr>
</tr>
</thead>
</thead>
Line 577: Line 577:
</li>
</li>
</li>
</li>
-
<li>2 samples of each of the 3 promoters: Added 5uL appropriate DNA and 0.5uL EcoRI and 0.5uL SpeI to 16uL MM</li>
+
<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 583: Line 583:
<tr>
<tr>
<th>Promoter MM 7x</th>
<th>Promoter MM 7x</th>
-
<th>Volume (uL)</th>
+
<th>Volume (µL)</th>
</tr>
</tr>
</thead>
</thead>
Line 614: Line 614:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
<li>Protocol: Ute Kothe</li>
<li>Protocol: Ute Kothe</li>
<li>Parts: J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1ak2</li>
<li>Parts: J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1ak2</li>
Line 711: Line 711:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
-
<li>Protocol: Biobasic Kit: Eluted with 30uL elution buffer</li>
+
<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>
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<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
<li>Protocol: Ute Kothe</li>
<li>Protocol: Ute Kothe</li>
<li>J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSb1Ak2</li>
<li>J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSb1Ak2</li>
-
<li>Separately added 2uL of appropriate DNA  and 0.2uL of Pfu Polymerase to 17.8 uL MM</li>\
+
<li>Separately added 2µL of appropriate DNA  and 0.2µL of Pfu Polymerase to 17.8µL MM</li>\
</ul>
</ul>
</div>
</div>
Line 779: Line 779:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<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>5 mL cultures, 5uL Amp. grown overnight at 37 degrees Celsius</li>
+
<li>5 mL 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 795: Line 795:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
<li>Protocol: Ute Kothe</li>
<li>Protocol: Ute Kothe</li>
-
<li>Mini Prep of (x2 of 35uL): J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002</li>
+
<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 35uL</li>
+
<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 814: Line 814:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
<li>Protocol: Ute Kothe</li>
<li>Protocol: Ute Kothe</li>
-
<li>Add 33uL DNA to 17uL MM</li>
+
<li>Add 33µL DNA to 17µL MM</li>
-
<li>17uL MM to promoter tubes</li>
+
<li>17µL MM to promoter tubes</li>
-
<li>33uL DNA to promoter tubes</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 37 degrees Celsius for 2hours</li>
+
<li>Incubated at 37°C for 2 hours</li>
-
<li>Heat killed at 65 degrees Celsius for 20minutes</li>
+
<li>Heat killed at 65°C for 20 minutes</li>
</ul>
</ul>
</div>
</div>
Line 871: Line 871:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
<li>Protocol: Ute Kothe</li>
<li>Protocol: Ute Kothe</li>
-
<li>Mini prep of (35uL EB): J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002</li>
+
<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 35uL</li>
+
<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 890: Line 890:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<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>50uL rxn</li>
+
<li>50µL rxn</li>
-
<li>Add 30uL DNA to 20uL MM</li>
+
<li>Add 30µL DNA to 20µL MM</li>
-
<li>Added 0.5uL EcroR1 & Pst1</li>
+
<li>Added 0.5µL EcroR1 & Pst1</li>
-
<li>Incubated 2hours at 37 degrees Celsius</li>
+
<li>Incubated 2 hours at 37°C</li>
-
<li>Heat killed 20minutes at 65 degrees Celsius</li>
+
<li>Heat killed 20 minutes at 65°C</li>
</ul>
</ul>
</div>
</div>
Line 938: Line 938:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<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: 55.6 degrees Celsius</li>
+
<li>PCR: Master mix created with: Annealing temperature: 55.6°C</li>
</ul>
</ul>
</div>
</div>
Line 1,011: Line 1,011:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
<li>Protocol: Ute Kothe</li>
<li>Protocol: Ute Kothe</li>
-
<li>Mini prep of (35uL EB): J06702_pSB1AK3, J23100_J61002, J23108_J61002 ,J23113_J61002, J23114_J61002</li>
+
<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 35uL</li>
+
<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 1,029: Line 1,029:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
<li>Protocol: Ute Kothe</li>
<li>Protocol: Ute Kothe</li>
-
<li>Mini prep of (35uL EB): J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002, J06702_PSB1AK2</li>
+
<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 35uL</li>
+
<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 1,047: Line 1,047:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<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, J23113 J61002</li>
<li>Parts: J23108_J61002, J23113 J61002</li>
-
<li>Eluted 50 uL</li>
+
<li>Eluted 50 µL</li>
</ul>
</ul>
</div>
</div>
Line 1,145: Line 1,145:
<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, 14uL MM, 0.5uL Spe1, 0.5uL Pst1</li>
+
<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 1,185: Line 1,185:
<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) 25uL DNA, 24uL MM, 0.5uL Spe1, 0.5uL Pst1</li>
+
<li>(Promoters) 25µL DNA, 24µL MM, 0.5µL Spe1, 0.5µL Pst1</li>
-
<li>(Insulin/Glucose) 25uL DNA, 24uL MM, 0.5uL EcoR1, 0.5uL Pst1</li>
+
<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 1,227: Line 1,227:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
<li>Protocol: Ute Kothe</li>
<li>Protocol: Ute Kothe</li>
</ul>
</ul>
Line 1,512: Line 1,512:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
<li>Protocol: Ute Kothe</li>
<li>Protocol: Ute Kothe</li>
</ul>
</ul>
Line 1,556: Line 1,556:
<td>5</td>
<td>5</td>
<td>J23100 mlc J06702</td>
<td>J23100 mlc J06702</td>
-
<td>PSB3C5</td>
+
<td>pSB3C5</td>
<td>50</td>
<td>50</td>
</tr>
</tr>
Line 1,562: Line 1,562:
<td>6</td>
<td>6</td>
<td>J23100 mlc J06702</td>
<td>J23100 mlc J06702</td>
-
<td>PSB3C5</td>
+
<td>pSB3C5</td>
<td>50</td>
<td>50</td>
</tr>
</tr>
Line 1,586: Line 1,586:
<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. Coli</li>
+
<li>Species: E. coli</li>
-
<li>Strain: DH5A</li>
+
<li>Strain: DH5α</li>
<li>Protocol: Ute Kothe</li>
<li>Protocol: Ute Kothe</li>
</ul>
</ul>

Revision as of 04:03, 19 June 2013

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

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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

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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

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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

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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

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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/mL
    J23108 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

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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.

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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

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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

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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

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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

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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

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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
  • 5 mL cultures, 5µL Amp. grown overnight at 37°C

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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

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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

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Agarose Gel of PCR 1%

  • Result: Successful

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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

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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 EcroR1 & 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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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Retrieved from "http://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april"