Team:Groningen/Notebook/31 August 2009
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The cloning strategy will be changed. First we will make a PCR2 and a PCR1.2 where F2-mut1rc plus Rev-mut2rc will generate a HmtA where we will add a prefix via a PCR with F1-REV. And than ready for cloning. But first we wait for a kit. | The cloning strategy will be changed. First we will make a PCR2 and a PCR1.2 where F2-mut1rc plus Rev-mut2rc will generate a HmtA where we will add a prefix via a PCR with F1-REV. And than ready for cloning. But first we wait for a kit. | ||
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+ | An aditional PCR started to get more megaprimer. | ||
===Metal Accumulation=== | ===Metal Accumulation=== |
Revision as of 10:41, 31 August 2009
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
HmtA
The cloning strategy will be changed. First we will make a PCR2 and a PCR1.2 where F2-mut1rc plus Rev-mut2rc will generate a HmtA where we will add a prefix via a PCR with F1-REV. And than ready for cloning. But first we wait for a kit.
An aditional PCR started to get more megaprimer.
Metal Accumulation
Vectors
Dry
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