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Team:Groningen/Notebook/13 July 2009
From 2009.igem.org
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===Transporters=== | ===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== | ==Dry== | ||
{{Team:Groningen/Notebook/Day/Footer}} | {{Team:Groningen/Notebook/Day/Footer}} | ||
Revision as of 11:19, 13 July 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106] were cut with EcoRI and SpeI.
- 0μL MQ
- 16μL plasmid in MQ ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106])
- 2μL Fast digest buffer
- 1μL EcoRI fast digest enzyme
- 1μL SpeI fast digest enzyme
-
- 6μL MQ
- 10μL plasmid in MQ ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 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
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