"
Team:Groningen/Notebook/17 September 2009
From 2009.igem.org
(Difference between revisions)
(New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== '''Planning''' :→ {{todo}} work out the wiki page for GVP :→ {{done}} make a doodle for presentation planning (1-19...) |
(→GVP Cluster) |
||
| Line 71: | Line 71: | ||
|x | |x | ||
|Yes (MvaI/XhoI) | |Yes (MvaI/XhoI) | ||
| + | |} | ||
| + | |||
| + | |||
| + | '''Restriction for Control and Assembly''' | ||
| + | |||
| + | The plasmids from the o.n. precultures of pArsR-GVP and pNL29 were cut with PstI/EcoRI and MvaI/XhoI to cut out the entire part between the pre- and suffix and wanted fragment. | ||
| + | |||
| + | {|cellpadding="2" cellspacing="1" border="4" | ||
| + | |'''Plasmid''' | ||
| + | |'''Amount μL''' | ||
| + | |'''MQ μL''' | ||
| + | |'''Fast digest buffer''' | ||
| + | |'''EcoRI fast digest enzyme''' | ||
| + | |'''PstI fast digest enzyme''' | ||
| + | |- | ||
| + | |pArsR-GVP no.1 | ||
| + | |16.0 | ||
| + | |x | ||
| + | |3.0 | ||
| + | |1.0 | ||
| + | |1.0 | ||
| + | |- | ||
| + | |pArsR-GVP no.2 | ||
| + | |16.0 | ||
| + | |x | ||
| + | |3.0 | ||
| + | |1.0 | ||
| + | |1.0 | ||
| + | |- | ||
| + | |pArsR-GVP no.2 | ||
| + | |16.0 | ||
| + | |x | ||
| + | |3.0 | ||
| + | |1.0 | ||
| + | |1.0 | ||
| + | |- | ||
| + | |'''Plasmid''' | ||
| + | |'''Amount μL''' | ||
| + | |'''MQ μL''' | ||
| + | |'''R-digest buffer''' | ||
| + | |'''MvaI digest enzyme''' | ||
| + | |'''XhoI digest enzyme''' | ||
| + | |- | ||
| + | |pNL29 | ||
| + | |16.0 | ||
| + | |x | ||
| + | |3.0 | ||
| + | |1.0 | ||
| + | |1.0 | ||
|} | |} | ||
Revision as of 08:43, 17 September 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
|
|---|
Wet
GVP Cluster
Planning
- → TODO work out the wiki page for GVP
- → DONE make a doodle for presentation planning (1-19 oct.)
- → TODO media attention
- → DONE place an ethics survey link on twitter
- → TODO clone pArsR-GVP into pSB2K3
- → TODO clone repeat out of GVP cluster
- → TODO make glycerol stocks of constructs
- → TODO enter info on part registry
Cultures
The overnight cultures with LB-amp100 medium of colonies E.coli TOP10 with pNL29 (3x), and pArsR-GVP (1-3) all showed expected growth of bacteria.
Plasmid isolation
Plasmid isolation was performed on the cultures of E.coli TOP10 containing the above mentioned plasmids with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™] Plasmid Miniprep Kit".
- From each tube 4mL of culture (8mL for pNL29) was collected in a 2.0mL cup (tubes from pArsR-GVP, GVP and pNL29 were combined), and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
- Plasmids were eluted with 30μL MQ and stored in the fridge
Concentrations
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| pArsR-GVP (pSB2K3, no.1) | 40.6 | 1.89 | 1.77 | x | Yes (EcoRI/PstI) |
| pArsR-GVP (pSB2K3, no.2) | 47.3 | 1.87 | 2.01 | x | Yes (EcoRI/PstI) |
| pArsR-GVP (pSB2K3, no.3) | 40.4 | 1.89 | 1.64 | x | Yes (EcoRI/PstI) |
| pNL29 | 26.4 | 2.10 | 1.64 | x | Yes (MvaI/XhoI) |
Restriction for Control and Assembly
The plasmids from the o.n. precultures of pArsR-GVP and pNL29 were cut with PstI/EcoRI and MvaI/XhoI to cut out the entire part between the pre- and suffix and wanted fragment.
| Plasmid | Amount μL | MQ μL | Fast digest buffer | EcoRI fast digest enzyme | PstI fast digest enzyme |
| pArsR-GVP no.1 | 16.0 | x | 3.0 | 1.0 | 1.0 |
| pArsR-GVP no.2 | 16.0 | x | 3.0 | 1.0 | 1.0 |
| pArsR-GVP no.2 | 16.0 | x | 3.0 | 1.0 | 1.0 |
| Plasmid | Amount μL | MQ μL | R-digest buffer | MvaI digest enzyme | XhoI digest enzyme |
| pNL29 | 16.0 | x | 3.0 | 1.0 | 1.0 |
Transporters
Metal Accumulation
Vectors
Dry
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
