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Team:Groningen/Notebook/31 August 2009
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(Difference between revisions)
(New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== '''Concentrations''' {|cellpadding="2" cellspacing="1" border="4" |'''Plasmid''' |'''Conc. ng/μL''' |'''260/280 |''...) |
(→GVP Cluster) |
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{|cellpadding="2" cellspacing="1" border="4" | {|cellpadding="2" cellspacing="1" border="4" | ||
|'''Plasmid''' | |'''Plasmid''' | ||
| + | |'''Amount μL''' | ||
| + | |'''MQ μL''' | ||
|'''Fast digest buffer''' | |'''Fast digest buffer''' | ||
|'''EcoRI fast digest enzyme''' | |'''EcoRI fast digest enzyme''' | ||
| Line 49: | Line 51: | ||
|'''SpeI fast digest enzyme''' | |'''SpeI fast digest enzyme''' | ||
|'''PstI fast digest enzyme''' | |'''PstI fast digest enzyme''' | ||
| - | |||
|- | |- | ||
|pSB1A2-pLacI-GVP | |pSB1A2-pLacI-GVP | ||
| - | | | + | |15.0 |
| - | | | + | |x |
| - | | | + | |3.0 |
| - | | | + | |1.0 |
| - | | | + | |x |
| + | |x | ||
| + | |1.0 | ||
|- | |- | ||
|pSB1A2-pBad/araC-GVP | |pSB1A2-pBad/araC-GVP | ||
| - | | | + | |4.0 |
| - | | | + | |12.0 |
| - | | | + | |3.0 |
| - | | | + | |1.0 |
| - | | | + | |x |
| + | |x | ||
| + | |1.0 | ||
|- | |- | ||
| - | |pSB1AC3- | + | |pSB1AC3-High |
| - | | | + | |6.0 |
| - | | | + | |9.0 |
| - | | | + | |3.0 |
| - | | | + | |1.0 |
| - | | | + | |x |
| + | |x | ||
| + | |1.0 | ||
|} | |} | ||
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Restriction was kept at 37C for 30 min. and put on ice until used for gel purification. | Restriction was kept at 37C for 30 min. and put on ice until used for gel purification. | ||
Revision as of 09:51, 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 | Amount μL | MQ μL | Fast digest buffer | EcoRI fast digest enzyme | XbaI fast digest enzyme | SpeI fast digest enzyme | PstI fast digest enzyme |
| pSB1A2-pLacI-GVP | 15.0 | x | 3.0 | 1.0 | x | x | 1.0 |
| pSB1A2-pBad/araC-GVP | 4.0 | 12.0 | 3.0 | 1.0 | x | x | 1.0 |
| pSB1AC3-High | 6.0 | 9.0 | 3.0 | 1.0 | x | x | 1.0 |
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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