From 2009.igem.org
Wet
GVP Cluster
- → TODO Restriction for Assembly
- → TODO Gel purification of plasmid/insert
- → TODO Ligation of GVP into vector pSB3K3
- → TODO Transformation of E.coli TOP10 cells (kan.)
- → TODO Test promoter/GVP constructs (grow precultures)
Restriction for Assembly
The vector pSB3K3 containing the low constitutive promoter was cut with PstI and SpeI to create correct ends for insert of GVP biobrick BBa_I750016, which was cut with XbaI and PstI.
- 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
Concentrations
Plasmid
| Conc. ng/μL
| 260/280
| 260/230
| -20 box (michael
| Restriction Control
|
pSB3K3-L (SpeI/PstI)
| ?
| ?
| ?
| ?
| ?
|
GVP (XbaI/PstI)
| ?
| ?
| ?
| ?
| ?
|
Restriction Control
Transporters
Metal Accumulation
Vectors
Dry