Team:Groningen/Notebook/13 July 2009
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The cups were incubated in a heat block at 37°C for 15 min. followed by adding 4μL 6x loading buffer for gel electroforese. | 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 electroforesis''' | ||
+ | |||
+ | 25μL of each sample was loaded on a 1% agarose gel with EtBr and a 1kb ladder was used (see picture). | ||
+ | |||
+ | [[]] [[Image:Generulers_1kb_marker_Fermentas.jpg]] | ||
+ | |||
+ | :→ From left to right: Empty Slot, 1kb Marker, | ||
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A standard kit for PCR-product purification was used for gel purification | 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 | + | * 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! | ||
===Transporters=== | ===Transporters=== |
Revision as of 14:13, 13 July 2009
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 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: Empty Slot, 1kb Marker,
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!
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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