Team:SDU-Denmark/labnotes6

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== Group: Retinal ==
== Group: Retinal ==
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==== ON of Top 10 E. coli cells with POT2 with NinaB ====
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Date: 16/8<br>
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Done by: Marie & Tommy<br>
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Methods:
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protocos:
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Notes:<br>
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100 mL ON culture was made, cells were grown at 37 C in LB media with Chloramphenicol.
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==== ON of Top 10 E. coli cells with POT2 with NinaB ====
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Date: 17/8<br>
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Done by: Marie & Tommy<br>
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Methods: Miniprep, Restriction digest, gel<br>
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Protocols:  [https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.2 MP1.2],  [https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1 RD1.1].
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Notes:<br>
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50 mL of elution buffer was used. DNA concentrations after pooling were measured on NanoDrop to 206,2 ng/microL.
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gel was run on produckt, showing recent miniprep compared to old miniprep produckt, showing uneven lengths. subsequent was performed.<br>
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[[Image:Team-sdu-denmark-Minipreb_product_of_POT2_with_NinaB_1,5_gel_red_loader__-4.jpg | 300px ]]
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==== Restriction digest on miniprep produckt (w. EcoRI) ====
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Date: 17/8<br>
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Done by: Marie & Tommy<br>
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Methods: Restriction digest, gel<br>
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Protocols: [https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.2 MP1.2], gel.
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Notes:<br>
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Due to the lack of old sample, restriction digest was performed using only 3,5 microL of miniprep produckt. 1,5 microL of H2O was added insted. EcoRI was used<br>
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The digest mix was incubated for 10 min at 37 C. A gel was run showing uncut new, cut new, uncut old and cut old miniprep product.<br>
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[[Image:Team-sdu-denmark-Minipreb_product_of_POT2_with_NinaB_1,5_gel_red_loader__-5.jpg |300px ]]
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=== PCR on POT2 with NinaB (New Primers) ===
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Start date: 20/8<br>
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Methods: PCR, Gel<br>
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Protocols: [https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP.1.1]
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==== PCR on POT2 with NinaB (New Primers) ====
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Date: 20/8<br>
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Done by: Marie & Tommy<br>
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Methods: ON<br>
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protocos:[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]
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Notes:<br>
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The new primers only contain the innermost restriction sites.<br>
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Meltting temp.: FWD: 68,1 C REV: 65,6 C<br>
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To get the optimal PCR temperatures a gradient PCR were run programed as:<br>
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<html>
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<head>
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  <meta content="text/html; charset=ISO-8859-1"
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http-equiv="content-type">
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  <title></title>
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</head>
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<body>
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<table style="text-align: left; width: 300px;" border="1"
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cellpadding="2" cellspacing="2">
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    <tr>
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      <td>PCR</td>
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      <td>Temp. (C)</td>
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      <td>Time (min)</td>
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    </tr>
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    <tr>
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      <td>Start</td>
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      <td>95</td>
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      <td>2</td>
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    </tr>
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    <tr>
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      <td>Denaturing</td>
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      <td>95</td>
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      <td>1</td>
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    </tr>
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    <tr>
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      <td>Anneling</td>
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      <td>Gradient</td>
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      <td>1</td>
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    </tr>
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    <tr>
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      <td>Elongation</td>
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      <td>72</td>
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      <td>4</td>
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    </tr>
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    <tr>
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      <td>End</td>
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      <td>72</td>
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      <td>5</td>
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    </tr>
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    <tr>
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      <td>Hold</td>
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      <td>4</td>
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      <td>indef.</td>
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    </tr>
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</table>
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<br>
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</body>
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</html>
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<br>
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The temperatur gradient were run from 60 to 70 C and the samples were run at these temperatures:<br>
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<html>
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<head>
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  <meta content="text/html; charset=ISO-8859-1"
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http-equiv="content-type">
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  <title></title>
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</head>
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<body>
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<table style="text-align: left; width: 300px;" border="1"
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cellpadding="2" cellspacing="2">
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    <tr>
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      <td>Sample</td>
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      <td>Colunm</td>
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      <td>Temp. C</td>
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    </tr>
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    <tr>
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      <td>1</td>
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      <td>1</td>
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      <td>59,9</td>
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    </tr>
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    <tr>
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      <td>2</td>
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      <td>3</td>
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      <td>60,7</td>
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    </tr>
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    <tr>
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      <td>3</td>
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      <td>5</td>
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      <td>62,7</td>
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    </tr>
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    <tr>
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      <td>4</td>
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      <td>7</td>
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      <td>65,4</td>
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    </tr>
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    <tr>
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      <td>5</td>
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      <td>9</td>
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      <td>67,9</td>
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    </tr>
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    <tr>
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      <td>6</td>
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      <td>11</td>
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      <td>69,6</td>
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    </tr>
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</table>
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<br>
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</body>
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</html><br>
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After the PRC the product were run on a gel<br>
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The gel picture shows that alle of the temperatures gave results.

Latest revision as of 22:21, 23 October 2010

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