Team:SDU-Denmark/labnotes14

From 2010.igem.org

(Difference between revisions)
(→Lab notes (10/11 - 10/17))
(→Lab notes (10/11 - 10/17))
 
(2 intermediate revisions not shown)
Line 11: Line 11:
<br>
<br>
Done By: Sheila and Louise<br>
Done By: Sheila and Louise<br>
-
Date: 10/13 2010<br>
+
Date: 11/10 2010<br>
Protocol: [https://2010.igem.org/Team:SDU-Denmark/protocols#TR1.1 TR1.1]<br>
Protocol: [https://2010.igem.org/Team:SDU-Denmark/protocols#TR1.1 TR1.1]<br>
Notes: <br>
Notes: <br>
Results: <br>
Results: <br>
Conclusion: <br>
Conclusion: <br>
 +
 +
== Retinal ==
 +
 +
=== Miniprep of K343005+DT in PSB1AK3 ===
 +
Date: 30/09<br>
 +
Done by: Marie<br>
 +
Methods: Miniprep<br>
 +
Protocols: MP1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.1]<br>
 +
Notes: <br>
 +
marie skriver dette...
 +
 +
=== Restriction digest on K343002 and PSB1C3 ===
 +
Date: 12/10<br>
 +
Done by: Tommy<br>
 +
Methods: Restriction digest<br>
 +
Protocols: RD1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1]<br>
 +
Notes: <br>
 +
EcoRI and SpeI were used, the producted was purifided on a gel<br>
 +
nanodrop:<br>
 +
PSB1C3: 41,6<br>
 +
K343002: 64,5<br>
 +
 +
=== Ligation of K343002 into PSB1C3 ===
 +
Date: 12/10<br>
 +
Done by: Marie & Christian<br>
 +
Methods: Ligation<br>
 +
Protocols: LG1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.1]<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 ===
 +
Date: 12/10<br>
 +
Done by: Tommy<br>
 +
Methods: Ligation<br>
 +
Protocols: LG1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.1]<br>
 +
Notes: <br>
 +
16 colonies and RFP control was selected from the colonie PCR. All of the colonies contain K343002 in PSB1C3<br>
 +
VF2 and VR primers was used<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&nbsp; 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;">3 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>
 +
 +
=== 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&nbsp; 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

Retrieved from "http://2010.igem.org/Team:SDU-Denmark/labnotes14"