"
Team:Hannover/Notebook/GFP
From 2014.igem.org
(Difference between revisions)
| Line 6: | Line 6: | ||
<html> | <html> | ||
<body> | <body> | ||
| - | <h1>Notebook / Locate GFP and the CBD</h1> | + | <h1><a href"https://2014.igem.org/Team:Hannover/Notebook">Notebook</a> / Locate GFP and the CBD</h1> |
<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> | <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> | ||
<table cellspacing="0" cellpadding="0" border="0" class="padtable"> | <table cellspacing="0" cellpadding="0" border="0" class="padtable"> | ||
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 |
