Team:SDU-Denmark/labnotes6
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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> | 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> | ||
[[Image:Team-sdu-denmark-Minipreb_product_of_POT2_with_NinaB_1,5_gel_red_loader__-5.jpg |400px ]] | [[Image:Team-sdu-denmark-Minipreb_product_of_POT2_with_NinaB_1,5_gel_red_loader__-5.jpg |400px ]] | ||
+ | |||
+ | === PCR on POT2 with NinaB (New Primers) === | ||
+ | Start date: 20/8<br> | ||
+ | Methods: PCR, Gel<br> | ||
+ | Protocols: CP.1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1] | ||
+ | |||
+ | ==== PCR on POT2 with NinaB (New Primers) ==== | ||
+ | Date: 20/8<br> | ||
+ | Done by: Marie & Tommy<br> | ||
+ | Methods: ON<br> | ||
+ | protocos:CP.1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1] | ||
+ | <br><br> | ||
+ | Notes:<br> | ||
+ | The new primers only contain the innermost restriction sites.<br> | ||
+ | Meltting temp.: FWD: 68,1 C REV: 65,6 C<br> | ||
+ | To get the optimal PCR temperatures a gradient PCR were run programed as:<br> | ||
+ | <html> | ||
+ | <head> | ||
+ | <meta content="text/html; charset=ISO-8859-1" | ||
+ | http-equiv="content-type"> | ||
+ | <title></title> | ||
+ | </head> | ||
+ | <body> | ||
+ | <table style="text-align: left; width: 100%;" border="1" | ||
+ | cellpadding="2" cellspacing="2"> | ||
+ | <tr> | ||
+ | <td>PCR</td> | ||
+ | <td>Temp. (C)</td> | ||
+ | <td>Time (min)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Start</td> | ||
+ | <td>95</td> | ||
+ | <td>2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Denaturing</td> | ||
+ | <td>95</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Anneling</td> | ||
+ | <td>Gradient</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Elongation</td> | ||
+ | <td>72</td> | ||
+ | <td>4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>End</td> | ||
+ | <td>72</td> | ||
+ | <td>5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Hold</td> | ||
+ | <td>4</td> | ||
+ | <td>indef.</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br> | ||
+ | </body> | ||
+ | </html> | ||
+ | <br> | ||
+ | The temperatur gradient were run from 60 to 70 C and the samples were run at these temperatures:<br> | ||
+ | <html> | ||
+ | <head> | ||
+ | <meta content="text/html; charset=ISO-8859-1" | ||
+ | http-equiv="content-type"> | ||
+ | <title></title> | ||
+ | </head> | ||
+ | <body> | ||
+ | <table style="text-align: left; width: 100%;" border="1" | ||
+ | cellpadding="2" cellspacing="2"> | ||
+ | <tr> | ||
+ | <td>Sample</td> | ||
+ | <td>Colunm</td> | ||
+ | <td>Temp. C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>59,9</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>3</td> | ||
+ | <td>60,7</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>5</td> | ||
+ | <td>62,7</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>7</td> | ||
+ | <td>65,4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>9</td> | ||
+ | <td>67,9</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>11</td> | ||
+ | <td>69,6</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br> | ||
+ | </body> | ||
+ | </html><br> | ||
+ | After the PRC the product were run on a gel<br> | ||
+ | |||
+ | The gel picture shows that alle of the temperatures gave results. | ||
+ | |||
+ | ==== Futher PCR on POT2 with NinaB (New Primers NO. 5) ==== | ||
+ | Date: 23/8<br> | ||
+ | Done by: Marie & Tommy<br> | ||
+ | Methods: PCR<br> | ||
+ | protocos:CP.1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1] | ||
+ | <br><br> | ||
+ | Notes:<br> | ||
+ | NinB2fw and NinaB2rv was used.<br> | ||
+ | PCR were run programed as:<br> | ||
+ | <html> | ||
+ | <head> | ||
+ | <meta content="text/html; charset=ISO-8859-1" | ||
+ | http-equiv="content-type"> | ||
+ | <title></title> | ||
+ | </head> | ||
+ | <body> | ||
+ | <table style="text-align: left; width: 100%;" border="1" | ||
+ | cellpadding="2" cellspacing="2"> | ||
+ | <tr> | ||
+ | <td>PCR</td> | ||
+ | <td>Temp. (C)</td> | ||
+ | <td>Time (min)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Start</td> | ||
+ | <td>95</td> | ||
+ | <td>2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Denaturing</td> | ||
+ | <td>95</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Anneling</td> | ||
+ | <td>67,9</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Elongation</td> | ||
+ | <td>72</td> | ||
+ | <td>4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>End</td> | ||
+ | <td>72</td> | ||
+ | <td>5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Hold</td> | ||
+ | <td>4</td> | ||
+ | <td>indef.</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br> | ||
+ | </body> | ||
+ | </html> | ||
+ | <br> | ||
+ | PCR product from gradient PCR (d. 20/8-10), tube no. 5, was used as template.<br> | ||
+ | The other tubes were pooled.<br> | ||
+ | |||
+ | === PCR on POT2 with NinaB (New Primers) === | ||
+ | Start date: 24/8<br> | ||
+ | Methods: Ligation, Competent cells, Transformation<br> | ||
+ | Protocols: LG1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.1], CC1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CC1.1], TR1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#TR1.1] | ||
+ | |||
+ | ==== DNA purification from PCR ==== | ||
+ | Date: 20/8<br> | ||
+ | Done by: Marie & Tommy<br> | ||
+ | Methods: DNA purification from PCR<br> | ||
+ | protocos:GFX purification from PCR - kit | ||
+ | <br><br> | ||
+ | Notes:<br> | ||
+ | One of the pooled tubes was eluted in 200µL, the others was eluted in 20µL<br> | ||
+ | 200µL nanodrop: 16,4 ng/µL<br> | ||
+ | 20µL nanodrop: 133,9ng/µL<br> | ||
+ | |||
+ | ==== Restriction Digest ==== | ||
+ | Date: 24/8<br> | ||
+ | Done by: Marie & Tommy<br> | ||
+ | Methods: Restriction Digest<br> | ||
+ | protocos:RD1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1] | ||
+ | <br><br> | ||
+ | Notes:<br> |
Revision as of 10:49, 1 September 2010