From 2009.igem.org
Wet
GVP Cluster
Restriction for Assembly
The vector containing GVP cluster was cut with EcoRI and XbaI to create correct ends.
- 6μL MQ
- 10μL plasmid in MQ
- 2μL Fast digest buffer
- 1μL EcoRI fast digest enzyme
- 1μL XbaI fast digest enzyme
The vectors containing promotors BBa_J23109, BBa_J23100 and BBa_J23106 were cut with EcoRI and SpeI.
- 0μL MQ
- 16μL plasmid in MQ (BBa_J23109 and BBa_J23106)
- 2μL Fast digest buffer
- 1μL EcoRI fast digest enzyme
- 1μL SpeI fast digest enzyme
-
- 6μL MQ
- 10μL plasmid in MQ (BBa_J23100)
- 2μL Fast digest buffer
- 1μL EcoRI fast digest enzyme
- 1μL SpeI fast digest enzyme
The cups were incubated in a heat block at 37°C for 15 min. followed by adding 4μL 6x loading buffer for gel electroforese.
Gel Purification of GVP
A standard kit for PCR-product purification was used for gel purification
- ..mg of gel containing the desired fragment was dissolved in ..μL binding buffer by heating to ..°C for ..min.
- the column was prepared with ..μL
Transporters
PCR program
| Temperature
| Time
|
Denaturing
| 95°
| 2.00 min
|
| Start Cycles 25X
|
Denaturing
| 95°
| 30 sec
|
Annealing
| 55°
| 20 sec
|
Elongation
| 72°
| 4.10 min
|
| End cycles
|
Final elongation
| 72°
| 10 min
|
Hold
| 4°
| Forever
|
Dry