Team:SDU-Denmark/labnotes14
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Protocols: LG1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.1]<br> | Protocols: LG1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.1]<br> | ||
Notes: <br> | Notes: <br> | ||
- | + | For each of the ligations three ligation mixtures were prepared. vector concentrations of 41,6ng/uL (PSB1C3) was used for mixture. Appropiate amount of insert was added to reach vector:insert ratios of 1:1, 1:2 and 1:3 respectively. <br> | |
+ | Ligation mixtures (B0015 in pSB3C5):<br> | ||
+ | <table style="text-align: left;" border="1" | ||
+ | cellpadding="2" cellspacing="2"> | ||
+ | <tr> | ||
+ | <td></td> | ||
+ | <td>L1</td> | ||
+ | <td>L2</td> | ||
+ | <td>L3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T4 ligase buffer</td> | ||
+ | <td>2uL</td> | ||
+ | <td>2uL</td> | ||
+ | <td>2uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T4 ligase</td> | ||
+ | <td>1uL</td> | ||
+ | <td>1uL</td> | ||
+ | <td>1uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3</td> | ||
+ | <td>1uL</td> | ||
+ | <td>1uL</td> | ||
+ | <td>1uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>K343002 </td> | ||
+ | <td>0.65uL</td> | ||
+ | <td>1.30uL</td> | ||
+ | <td>1.96uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>H<small>2</small>0</td> | ||
+ | <td>34.35uL</td> | ||
+ | <td>33,70uL</td> | ||
+ | <td>33.04uL</td> | ||
+ | </tr> | ||
+ | </table><br> | ||
+ | The samples was incubated at 17C ON at used for transformation<br> | ||
=== Transformations of K343002 in PSB1C3 into top10 E. Coli === | === Transformations of K343002 in PSB1C3 into top10 E. Coli === | ||
Line 114: | Line 155: | ||
</tr> | </tr> | ||
</table> | </table> | ||
+ | |||
+ | === Coloni PCR on transformations of K343002 in PSB1C3 === | ||
+ | Date: 30/09<br> | ||
+ | Done by: Tommy<br> | ||
+ | Methods: PCR<br> | ||
+ | Protocols: CP1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3]<br> | ||
+ | Notes: <br> | ||
+ | Because the first coloni PCR of the transformants didn't give us the the right pieces 32 new colonies where chosen:<br> | ||
+ | 1-16: ninaB in PSB1C3<br> | ||
+ | |||
+ | VF2 and VR used as primers in the TAQ PCR:<br> | ||
+ | |||
+ | PCR Program:<br> | ||
+ | <table style="text-align: left;" border="1" cellpadding="2" | ||
+ | cellspacing="2"> | ||
+ | <tr> | ||
+ | <td style="vertical-align: top;">Start<br> | ||
+ | </td> | ||
+ | <td style="vertical-align: top;">94 C<br> | ||
+ | </td> | ||
+ | <td style="vertical-align: top;">2 min<br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="vertical-align: top;">Denaturing<br> | ||
+ | </td> | ||
+ | <td style="vertical-align: top;">94 C<br> | ||
+ | </td> | ||
+ | <td style="vertical-align: top;">1 min<br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="vertical-align: top;">Annealing<br> | ||
+ | </td> | ||
+ | <td style="vertical-align: top;">55 C<br> | ||
+ | </td> | ||
+ | <td style="vertical-align: top;">1 min<br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="vertical-align: top;">Elongation<br> | ||
+ | </td> | ||
+ | <td style="vertical-align: top;">72 C<br> | ||
+ | </td> | ||
+ | <td style="vertical-align: top;">2:30 min<br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="vertical-align: top;">Goto2<br> | ||
+ | </td> | ||
+ | <td style="vertical-align: top;">rep<br> | ||
+ | </td> | ||
+ | <td style="vertical-align: top;">29x<br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="vertical-align: top;">End<br> | ||
+ | </td> | ||
+ | <td style="vertical-align: top;">72 C<br> | ||
+ | </td> | ||
+ | <td style="vertical-align: top;">2:30 min<br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="vertical-align: top;">Hold<br> | ||
+ | </td> | ||
+ | <td style="vertical-align: top;">4 C<br> | ||
+ | </td> | ||
+ | <td style="vertical-align: top;"><br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | Colonies 1, 2, 3 and 4 was selected for minipreps and freezing cultures. |
Latest revision as of 16:32, 16 October 2010