Team:SDU-Denmark/labnotes9
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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> | ||
+ | === Miniprep and controle digestion of PS in pSB1C3 and pSB1AK3 === | ||
+ | '''Date:''' 9/13 2010<Br> | ||
+ | '''Done By:''' Maria and Lc<Br> | ||
+ | '''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> | ||
+ | '''Notes:'''<br> | ||
+ | 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> | ||
+ | the 2 minipreps of each coloni are diluted in 20uL and 50uL respectively.<br> | ||
+ | Restriction mixture x4:<br> | ||
+ | <table style="text-align: left;" border="1" | ||
+ | cellpadding="2" cellspacing="2"> | ||
+ | <tr> | ||
+ | <td>H<small>2</small>O</td> | ||
+ | <td>48uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>FD green buffer</td> | ||
+ | <td>8uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoRI</td> | ||
+ | <td>4uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PstI</td> | ||
+ | <td>4uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Plasmid</td> | ||
+ | <td>4x5uL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br> | ||
+ | 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> | ||
+ | Loading scheme:<br> | ||
+ | <table style="text-align: left;" border="1" | ||
+ | cellpadding="2" cellspacing="2"> | ||
+ | <tr> | ||
+ | <td>Lane</td> | ||
+ | <td>Sample</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>marker</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>digested plasmid from col. B</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>undigested plasmid from col. B</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>digested plasmid from col. F</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>undigested plasmid from col. F</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>digested plasmid from col. H</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7</td> | ||
+ | <td>undigested plasmid from col. H</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8</td> | ||
+ | <td>digested plasmid from col. C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9</td> | ||
+ | <td>undigested plasmid from col. C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10</td> | ||
+ | <td>marker</td> | ||
+ | </tr> | ||
+ | </table><br> | ||
+ | concentrations of miniprep was measured on nanodrop.<br><br> | ||
+ | '''Results:'''<br> | ||
+ | nanodrop:<br> | ||
+ | <table style="text-align: left;" border="1" | ||
+ | cellpadding="2" cellspacing="2"> | ||
+ | <tr> | ||
+ | <td>sample</td> | ||
+ | <td>conc. ng/uL</td> | ||
+ | <td>260/280</td> | ||
+ | <td>260/230</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PS i pSB1C3 B1</td> | ||
+ | <td>232.86</td> | ||
+ | <td>1.91</td> | ||
+ | <td>2.17</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PS i pSB1C3 B2</td> | ||
+ | <td>134.5</td> | ||
+ | <td>1.93</td> | ||
+ | <td>2.17</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PS i pSB1C3 F1</td> | ||
+ | <td>229.32</td> | ||
+ | <td>1.9</td> | ||
+ | <td>2.18</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PS i pSB1C3 F2</td> | ||
+ | <td>144.11</td> | ||
+ | <td>1.92</td> | ||
+ | <td>2.15</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PS i pSB1C3 H1</td> | ||
+ | <td>190.88</td> | ||
+ | <td>1.91</td> | ||
+ | <td>2.2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PS i pSB1C3 H2</td> | ||
+ | <td>137.54</td> | ||
+ | <td>1.89</td> | ||
+ | <td>2.15</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PS + double terminator in pSB1AK3 C1</td> | ||
+ | <td>227.6</td> | ||
+ | <td>1.91</td> | ||
+ | <td>2.2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PS + double terminator in pSB1AK3 C2</td> | ||
+ | <td>138.57</td> | ||
+ | <td>1.92</td> | ||
+ | <td>2.18</td> | ||
+ | </tr> | ||
+ | </table><br><br> | ||
+ | '''Analysis:'''<br> | ||
+ | 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> | ||
+ | --[[User:Tipi|Tipi]] 14:44, 26 September 2010 (UTC)<br><br> |
Revision as of 14:44, 26 September 2010