Team:Hannover/Notebook/GFP

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<h1><a href"https://2014.igem.org/Team:Hannover/Notebook">Notebook</a> / Locate GFP and the CBD</h1>
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<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.<br><br>ONC = overnight culture ; CBD = Cellulose binding domain ; IPG = Institute for plant genetics ; OD = optical density ;  PCR = Polymerase chain reaction ;  RT = room temperature ; T4MBP = Top 4 metal binding protein ; EMP = Exponential Megapriming PCR ; GFP = Green fluorescent protein</p>
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<table cellspacing="0" cellpadding="0" border="0" class="padtable">
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<tr class="todd">
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<td><h4>date</h4></td>
 +
<td><h4>coworkers</h4></td>
 +
<td><h4>lab</h4></td>
 +
<td><h4>activity</h4></td>
 +
<td><h4>short summary</h4></td>
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</tr>
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<tr class="teven">
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<td>09-Sept-2014 </td>
 +
<td>Fabian, Anke </td>
 +
<td>Biophysics </td>
 +
<td>confocal microscopy </td>
 +
<td>
 +
microscopy of transformed
 +
<i>B. sinuspersici</i>
 +
and
 +
<i>N. tabacum</i>
 +
(04-Sept-2014)
 +
</td>
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</tr>
 +
<tr>
 +
<td>04-Sept-2014 </td>
 +
<td>Fabian </td>
 +
<td>Botany </td>
 +
<td>transient plant transformation </td>
 +
<td>
 +
<i>A. tumefaciens</i>
 +
mediated transformation of
 +
<i>B. sinuspersici</i>
 +
(vacuum ilfiltration) and
 +
<i>N. tabacum</i>
 +
(leaf infiltration): used constructs: pORE_Expa~GFP~CBD, pEarley103_GFP, pEarley103_Plasmamembranemarker
 +
</td>
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</tr>
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<tr class="teven">
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<td>01-Sept-2014 </td>
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<td>Fabian, Steffen, Anke, Andreas </td>
 +
<td>Biophysics </td>
 +
<td>confocal microscopy </td>
 +
<td>
 +
microscopy of transformed
 +
<i>B. sinuspersici</i>
 +
and
 +
<i>N. tabacum</i>
 +
(transformation date: 29-Aug-2014)
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>29-Aug-2014 </td>
 +
<td>Fabian </td>
 +
<td>Botany </td>
 +
<td>transient plant transformation </td>
 +
<td>
 +
<i>A. tumefaciens</i>
 +
mediated transformation of
 +
<i>B. sinuspersici</i>
 +
(vacuum infiltration) and
 +
<i>N. tabacum</i>
 +
(leaf infiltration): used constructs: pORE_Expa~GFP~CBD, pEarley103_GFP, pEarley103_Plasmamembranemarker
 +
</td>
 +
</tr>
 +
<tr class="teven">
 +
<td>22-Aug-2014 </td>
 +
<td>Fabian </td>
 +
<td>Botany </td>
 +
<td>colony-PCR </td>
 +
<td>
 +
colony-PCR using
 +
<i>E. coli</i>
 +
colonies (20-Aug-2014) and primer 11 (Botany) and 1261: 15 positive clones/ ONC of 3 of 15 positives clones
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>21-Aug-2014 </td>
 +
<td>Fabian </td>
 +
<td>Botany </td>
 +
<td>cloning Expa~GFP~CBD into pORE </td>
 +
<td>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 </td>
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</tr>
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<tr class="teven">
 +
<td>19-Aug-2014 </td>
 +
<td>Fabian </td>
 +
<td>Botany </td>
 +
<td>Phusion PCR </td>
 +
<td>Phusion PCR using Expa~GFP~CBD-sequence from EMP as template </td>
 +
</tr>
 +
<tr>
 +
<td>08-Aug-2014 </td>
 +
<td>Andreas </td>
 +
<td>IPG </td>
 +
<td>plasmid-isolation </td>
 +
<td>isolation of pORE-E3 for future cloning and EMP_in_pMA_Colony1 for sequencing </td>
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</tr>
 +
<tr class="teven">
 +
<td>07-Aug-2014 </td>
 +
<td>Andreas </td>
 +
<td>IPG </td>
 +
<td>colony-PCR </td>
 +
<td>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) </td>
 +
</tr>
 +
<tr>
 +
<td>05-Aug-2014 </td>
 +
<td>Andreas </td>
 +
<td>IPG </td>
 +
<td>cellulose-bound-protein GFP_in_pMA_EMP2 </td>
 +
<td>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 </td>
 +
</tr>
 +
<tr  class="teven">
 +
<td>25-July-2014 </td>
 +
<td>Anke </td>
 +
<td>IPG </td>
 +
<td>colony-PCR of EMP2-transformation.v2 </td>
 +
<td>colony-PCR with primer 1011 and 625 (IPG) and 10 new colonies: poor results </td>
 +
</tr>
 +
<tr>
 +
<td>23-July-2014 </td>
 +
<td>Anke </td>
 +
<td>IPG </td>
 +
<td>colony-PCR of EMP2-transformation.v1 </td>
 +
<td>colony-PCR with primer 1011 and 625 (IPG): poor results </td>
 +
</tr>
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<tr class="teven">
 +
<td>22-July-2014 </td>
 +
<td>Anke </td>
 +
<td>IPG </td>
 +
<td>
 +
transformation of
 +
<i>E. coli </i>
 +
with EMP2
 +
</td>
 +
<td>transformation of XL1-Blue Competent Cells </td>
 +
</tr>
 +
<tr>
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<td>21-July-2014 </td>
 +
<td>
 +
Anke, Andreas
 +
</td>
 +
<td>IPG </td>
 +
<td>EMP2 GFP </td>
 +
<td>screening the functional amount of megaprimer with and without F1: 20 ng megaprimer + F1 + R2 </td>
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</tr>
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<tr class="teven">
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<td>18-July-2014 </td>
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<td>Anke </td>
 +
<td>IPG </td>
 +
<td>EMP1 GFP </td>
 +
<td>EMP1 with F1, R2 and GFP-vector to generate megaprimer: fine result </td>
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</tr>
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</tbody>
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</table>
<p id="Fusszeile"><br><br><br><br><img src="https://static.igem.org/mediawiki/2014/6/6a/Hannover_20141010Fusszeile2.jpg" width="980px" style="float:right" /></p>
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</body>
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Latest revision as of 13:30, 17 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.

ONC = overnight culture ; CBD = Cellulose binding domain ; IPG = Institute for plant genetics ; OD = optical density ; PCR = Polymerase chain reaction ; RT = room temperature ; T4MBP = Top 4 metal binding protein ; EMP = Exponential Megapriming PCR ; GFP = Green fluorescent protein

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





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