Team:SDU-Denmark/labnotes9

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(Insertion of PS in pSB1C3 and pSB1AK3)
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The transformation was successfull and colonies was selected and used in coloni PCR.<br>
The transformation was successfull and colonies was selected and used in coloni PCR.<br>
--[[User:Tipi|Tipi]] 13:41, 26 September 2010 (UTC)<br><br>
--[[User:Tipi|Tipi]] 13:41, 26 September 2010 (UTC)<br><br>
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=== Miniprep and controle digestion of PS in pSB1C3 and pSB1AK3 ===
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'''Date:''' 9/13 2010<Br>
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'''Done By:''' Maria and Lc<Br>
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'''Protocol:'''[https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.2 MP1.2][https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1 RD1.1]<br>
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'''Notes:'''<br>
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Coloni B, F and H of PS in pSB1C3 and coloni C of PS + double terminator in pSB1AK3 was miniprep'ed according to prtocol. To ensure that the plasmids contains the insert as expected, the miniprep is cut with EcoRI and PstI. <br>
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the 2 minipreps of each coloni are diluted in 20uL and 50uL respectively.<br>
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Restriction mixture x4:<br>
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<table style="text-align: left;" border="1"
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cellpadding="2" cellspacing="2">
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    <tr>
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      <td>H<small>2</small>O</td>
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      <td>48uL</td>
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    </tr>
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    <tr>
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      <td>FD green buffer</td>
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      <td>8uL</td>
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    </tr>
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    <tr>
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      <td>EcoRI</td>
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      <td>4uL</td>
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    </tr>
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    <tr>
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      <td>PstI</td>
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      <td>4uL</td>
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    </tr>
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    <tr>
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      <td>Plasmid</td>
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      <td>4x5uL</td>
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    </tr>
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</table>
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<br>
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The digested products are loaded onto a 1% agarose gel. 2.5uL of undigested plasmid are used as controle. Gene ruler DNA ladder mix are used as marker<br>
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Loading scheme:<br>
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<table style="text-align: left;" border="1"
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cellpadding="2" cellspacing="2">
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    <tr>
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      <td>Lane</td>
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      <td>Sample</td>
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    </tr>
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    <tr>
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      <td>1</td>
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      <td>marker</td>
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    </tr>
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    <tr>
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      <td>2</td>
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      <td>digested plasmid from col. B</td>
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    </tr>
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    <tr>
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      <td>3</td>
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      <td>undigested plasmid from col. B</td>
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    </tr>
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    <tr>
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      <td>4</td>
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      <td>digested plasmid from col. F</td>
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    </tr>
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    <tr>
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      <td>5</td>
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      <td>undigested plasmid from col. F</td>
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    </tr>
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    <tr>
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      <td>6</td>
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      <td>digested plasmid from col. H</td>
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    </tr>
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    <tr>
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      <td>7</td>
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      <td>undigested plasmid from col. H</td>
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    </tr>
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    <tr>
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      <td>8</td>
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      <td>digested plasmid from col. C</td>
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    </tr>
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    <tr>
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      <td>9</td>
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      <td>undigested plasmid from col. C</td>
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    </tr>
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    <tr>
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      <td>10</td>
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      <td>marker</td>
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    </tr>
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</table><br>
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concentrations of miniprep was measured on nanodrop.<br><br>
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'''Results:'''<br>
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nanodrop:<br>
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<table style="text-align: left;" 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>conc. ng/uL</td>
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      <td>260/280</td>
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      <td>260/230</td>
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    </tr>
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    <tr>
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      <td>PS i pSB1C3 B1</td>
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      <td>232.86</td>
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      <td>1.91</td>
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      <td>2.17</td>
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    </tr>
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    <tr>
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      <td>PS i pSB1C3 B2</td>
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      <td>134.5</td>
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      <td>1.93</td>
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      <td>2.17</td>
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    </tr>
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    <tr>
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      <td>PS i pSB1C3 F1</td>
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      <td>229.32</td>
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      <td>1.9</td>
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      <td>2.18</td>
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    </tr>
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    <tr>
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      <td>PS i pSB1C3 F2</td>
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      <td>144.11</td>
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      <td>1.92</td>
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      <td>2.15</td>
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    </tr>
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    <tr>
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      <td>PS i&nbsp;pSB1C3 H1</td>
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      <td>190.88</td>
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      <td>1.91</td>
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      <td>2.2</td>
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    </tr>
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    <tr>
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      <td>PS i pSB1C3 H2</td>
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      <td>137.54</td>
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      <td>1.89</td>
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      <td>2.15</td>
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    </tr>
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    <tr>
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      <td>PS + double terminator in pSB1AK3 C1</td>
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      <td>227.6</td>
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      <td>1.91</td>
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      <td>2.2</td>
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    </tr>
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    <tr>
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      <td>PS + double terminator in pSB1AK3 C2</td>
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      <td>138.57</td>
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      <td>1.92</td>
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      <td>2.18</td>
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    </tr>
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</table><br><br>
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'''Analysis:'''<br>
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In the lanes containing the digested pSB1C3 plasmids bands at app. 2000bp are observed corresponding to PS. The lane containing digested pSB1AK3 plasmids shows a band at app. 2100bp corresponding to the size of PS and the double terminator. Miniprep of coloni B, F and C are sent to sequentation.<br>
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--[[User:Tipi|Tipi]] 14:44, 26 September 2010 (UTC)<br><br>

Revision as of 14:44, 26 September 2010