From 2009.igem.org
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.
Metal Accumulation
Vectors
Dry