Team:Groningen/Notebook/13 July 2009
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 electroforesis.
Gel electroforesis
25μL of each sample was loaded on a 1% agarose gel with EtBr and a 1kb ladder was used (see picture).
- → From left to right: 1kb Marker, Empty Slot, BBa_J23109, Empty Slot, BBa_J23100, Empty Slot, BBa_J23106, Empty Slot, GVP, Empty Slot
- → The promotor parts (50bp) were not visible on gel after a run at 100V for 25 min. A second gel run for 10 min. at 100V showed the same result for promotor BBa_J23100, but had a vage outline of the correct fragment size (gel not shown). Purification of this part of the gel would yield a far too low concentration, and was therefor not attempted.
Gel Purification of GVP
A standard kit for PCR-product purification was used for gel purification
- 120mg of gel containing the desired fragment was dissolved in 500μL binding buffer by heating to 60°C for 10 min.
- the column was prepared with 500μL Preparation Solution and centrifuged for 1 min.
- dissolved gel was transfered to the column and centrifuged for 1 min.
- column was washed with 750μL Wash Solution and centrifuged twice for 1 min.
- 15μL MQ was applied to the column, incubated for 3 min. at room temperature and centrifuged for 1 min. at full speed.
Concentration of GVP-vector
The concentration of isolated cut vector of GVP with EcoRI and XbaI was determined with the use of a nano-drop.
GVP-vector eluted in MQ
- 4.3 ng/μL
- 1.42 (260/280)
- 0.60 (260/230)
- → After the low concentration was determined, the cup was disposed of because a higher concentration is required!
Inoculation of TY-medium
TY-medium (5mL) was inoculated with a tip of glycerol stocks:
- GVP no.1
- BBa_J23109
- BBa_J23100
- BBa_J23106
and 5μL ampicilin (1000x). The tubes were placed in a 37°C shacker for over night incubation at 200 rpm.
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
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