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Team:Hannover/Notebook/GFP
From 2014.igem.org
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<h1>Notebook / Locate GFP and the CBD</h1> | <h1>Notebook / Locate GFP and the CBD</h1> | ||
| - | <p class="text">Here we list all the steps that were required to locate the CBD within the cell using GFP as a marker.</p> | + | <p class="text">Here we list all the steps that were required to locate the cellulose binding domain (CBD) within the cell using GFP as a marker.</p> |
<table cellspacing="0" cellpadding="0" border="0" class="padtable"> | <table cellspacing="0" cellpadding="0" border="0" class="padtable"> | ||
<tr class="todd"> | <tr class="todd"> | ||
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<tr class="teven"> | <tr class="teven"> | ||
<td>01-Sept-2014 </td> | <td>01-Sept-2014 </td> | ||
| - | <td>Fabian, Steffen, Anke, | + | <td>Fabian, Steffen, Anke, Andreas </td> |
<td>Biophysics </td> | <td>Biophysics </td> | ||
<td>confocal microscopy </td> | <td>confocal microscopy </td> | ||
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<tr> | <tr> | ||
<td>08-Aug-2014 </td> | <td>08-Aug-2014 </td> | ||
| - | <td> | + | <td>Andreas </td> |
<td>IPG </td> | <td>IPG </td> | ||
<td>plasmid-isolation </td> | <td>plasmid-isolation </td> | ||
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<tr class="teven"> | <tr class="teven"> | ||
<td>07-Aug-2014 </td> | <td>07-Aug-2014 </td> | ||
| - | <td> | + | <td>Andreas </td> |
<td>IPG </td> | <td>IPG </td> | ||
<td>colony-PCR </td> | <td>colony-PCR </td> | ||
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<tr> | <tr> | ||
<td>05-Aug-2014 </td> | <td>05-Aug-2014 </td> | ||
| - | <td> | + | <td>Andreas </td> |
<td>IPG </td> | <td>IPG </td> | ||
<td>cellulose-bound-protein GFP_in_pMA_EMP2 </td> | <td>cellulose-bound-protein GFP_in_pMA_EMP2 </td> | ||
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<td>21-July-2014 </td> | <td>21-July-2014 </td> | ||
<td> | <td> | ||
| - | Anke, | + | Anke, Andreas |
</td> | </td> | ||
<td>IPG </td> | <td>IPG </td> | ||
Revision as of 04:25, 13 October 2014
Notebook / Locate GFP and the CBD
Here we list all the steps that were required to locate the cellulose binding domain (CBD) within the cell using GFP as a marker.
date |
coworkers |
lab |
activity |
short summary |
| 09-Sept-2014 | Fabian, Anke | Biophysics | confocal microscopy | microscopy of transformed B. sinuspersici and N. tabacum (04-Sept-2014) |
| 04-Sept-2014 | Fabian | Botany | transient plant transformation | A. tumefaciens mediated transformation of B. sinuspersici (vacuum ilfiltration) and N. tabacum (leaf infiltration): used constructs: pORE_Expa~GFP~CBD, pEarley103_GFP, pEarley103_Plasmamembranemarker |
| 01-Sept-2014 | Fabian, Steffen, Anke, Andreas | Biophysics | confocal microscopy | microscopy of transformed B. sinuspersici and N. tabacum (transformation date: 29-Aug-2014) |
| 29-Aug-2014 | Fabian | Botany | transient plant transformation | A. tumefaciens mediated transformation of B. sinuspersici (vacuum infiltration) and N. tabacum (leaf infiltration): used constructs: pORE_Expa~GFP~CBD, pEarley103_GFP, pEarley103_Plasmamembranemarker |
| 22-Aug-2014 | Fabian | Botany | colony-PCR | colony-PCR using E. coli colonies (20-Aug-2014) and primer 11 (Botany) and 1261: 15 positive clones/ ONC of 3 of 15 positives clones |
| 21-Aug-2014 | Fabian | Botany | cloning Expa~GFP~CBD into pORE | purification of PCR-product (19-Aug-2014)/ restriction digest of PCR-product and pORE E3 2x35S with BamHI and MluI/ gelextraction of digested vector and PCR-product/ ligation for 1 h/ transformation of XL1-Blue Competent Cells via heat-shock/ selection on kanamycin |
| 19-Aug-2014 | Fabian | Botany | Phusion PCR | Phusion PCR using Expa~GFP~CBD-sequence from EMP as template |
| 08-Aug-2014 | Andreas | IPG | plasmid-isolation | isolation of pORE-E3 for future cloning and EMP_in_pMA_Colony1 for sequencing |
| 07-Aug-2014 | Andreas | IPG | colony-PCR | although colony-PCR (F1+R1) confirmed insertion of GFP: another primer set (1101 and 625, IPG) targeting the backbone of pMA did not bind in the colony, but showed an unexpected amplificate length of ~1200 bp in the negative control (expected for pMA: ~1600 bp) |
| 05-Aug-2014 | Andreas | IPG | cellulose-bound-protein GFP_in_pMA_EMP2 | repetition of EMP-PCR(2) reaction mix no. 2 (protocol of Anke): gel confirmed the correct fragment length/ purification using the „Wizard-Kit“/ phosphorylation/ ligation overnight according to Anke's protocol |
| 25-July-2014 | Anke | IPG | colony-PCR of EMP2-transformation.v2 | colony-PCR with primer 1011 and 625 (IPG) and 10 new colonies: poor results |
| 23-July-2014 | Anke | IPG | colony-PCR of EMP2-transformation.v1 | colony-PCR with primer 1011 and 625 (IPG): poor results |
| 22-July-2014 | Anke | IPG | transformation of E. coli with EMP2 | transformation of XL1-Blue Competent Cells |
| 21-July-2014 | Anke, Andreas | IPG | EMP2 GFP | screening the functional amount of megaprimer with and without F1: 20 ng megaprimer + F1 + R2 |
| 18-July-2014 | Anke | IPG | EMP1 GFP | EMP1 with F1, R2 and GFP-vector to generate megaprimer: fine result |
