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Team:Groningen/Notebook/31 August 2009
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(New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== '''Concentrations''' {|cellpadding="2" cellspacing="1" border="4" |'''Plasmid''' |'''Conc. ng/μL''' |'''260/280 |''...)
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(New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== '''Concentrations''' {|cellpadding="2" cellspacing="1" border="4" |'''Plasmid''' |'''Conc. ng/μL''' |'''260/280 |''...)
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Revision as of 09:00, 31 August 2009
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Wet
GVP Cluster
Concentrations
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| pSB1A2-pLacI-GVP | 41.2 | 1.85 | 2.18 | ? | Yes (EcoRI/PstI) |
| pSB1A2-pBad/araC-GVP | 421.7 | 1.84 | 2.34 | ? | Yes (EcoRI/PstI) |
| pSB1AC3-H | 2.43.1 | 1.86 | 2.17 | All Used | Yes (Glycerol Stock) |
Restriction for Assembly
The vector pSB1A2 containing the pLacI/GVP and pBad/araC/GVP composite parts were cut with PstI and EcoRI to create correct ends for insert into pSB1AC3, which was also cut with EcoRI and PstI.
| Plasmid | Fast digest buffer | EcoRI fast digest enzyme | XbaI fast digest enzyme | SpeI fast digest enzyme | PstI fast digest enzyme | Restriction Control |
| pSB1A2-pLacI-GVP | 41.2 | 1.85 | 2.18 | ? | Yes (EcoRI/PstI) | |
| pSB1A2-pBad/araC-GVP | 421.7 | 1.84 | 2.34 | ? | Yes (EcoRI/PstI) | |
| pSB1AC3-H | 2.43.1 | 1.86 | 2.17 | All Used | Yes (Glycerol Stock) |
- 16μL pSB3K3-L in MQ (0.8μg)
- 2μL Fast digest buffer
- 1μL PstI fast digest enzyme
- 1μL SpeI fast digest enzyme
--
- 10μL plasmid-GVP in MQ (?μg)
- 6μL MQ (end volume of 20μL)
- 2μL Fast digest buffer
- 1μL PstI fast digest enzyme
- 1μL SpeI/XbaI fast digest enzyme
Restriction was kept at 37C for 30 min. and put on ice until used for gel purification.
Purification
A "Agarose Gel DNA Extraction Kit" Standard Protocol from Roche Applied Science.
- → In step ten, 30μL MQ was added to the dry pellet and incubated at room temperature.
Transporters
Metal Accumulation
Vectors
Dry
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