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Team:Groningen/Notebook/17 July 2009
From 2009.igem.org
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===Transporters=== | ===Transporters=== | ||
| + | {| | ||
| + | | | ||
| + | <!--Tabel 1 hier--> | ||
| + | |||
| + | {| border="1" | ||
| + | |+ '''MasterMix3''' | ||
| + | ! Component !! Mg/K | ||
| + | |- | ||
| + | ! MQ | ||
| + | | 28.5 uL | ||
| + | |- | ||
| + | ! F mut1 | ||
| + | | 2 uL | ||
| + | |- | ||
| + | ! R mut2 | ||
| + | | 2 uL | ||
| + | |- | ||
| + | ! dNTP | ||
| + | | 2 uL | ||
| + | |- | ||
| + | ! MgCl | ||
| + | | 3 uL | ||
| + | |- | ||
| + | ! taq buffer KCl | ||
| + | | 5 uL | ||
| + | |- | ||
| + | ! taq buffer (NH)SO | ||
| + | | 5 uL | ||
| + | |- | ||
| + | ! taq polymerase | ||
| + | | 2.5 uL | ||
| + | |- | ||
| + | |} | ||
| + | |||
| + | |width="10"| | ||
| + | | | ||
| + | <!--Tabel 2 hier--> | ||
| + | {| | ||
| + | ! PCR 3 program | ||
| + | ! Temperature | ||
| + | ! Time | ||
| + | |- | ||
| + | |Denaturing | ||
| + | |95° | ||
| + | |2.00 min | ||
| + | |- | ||
| + | | | ||
| + | |Start Cycles 25X | ||
| + | |- | ||
| + | |Denaturing | ||
| + | |95° | ||
| + | |30 sec | ||
| + | |- | ||
| + | |Annealing | ||
| + | |55° | ||
| + | |20 sec | ||
| + | |- | ||
| + | |Elongation | ||
| + | |72° | ||
| + | |2.10 min | ||
| + | |- | ||
| + | | | ||
| + | |End cycles | ||
| + | |- | ||
| + | |Final elongation | ||
| + | |72° | ||
| + | |10 min | ||
| + | |- | ||
| + | |Hold | ||
| + | |4° | ||
| + | |Forever | ||
| + | |} | ||
| + | |width="10"| | ||
| + | | | ||
| + | <!--Tabel 3 hier--> | ||
| + | {| | ||
| + | [[Image:PCR15-7-19.jpg]] | ||
| + | |} | ||
| + | |} | ||
===Metal Accumulation=== | ===Metal Accumulation=== | ||
Revision as of 10:24, 17 July 2009
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Wet
GVP Cluster
Discussion:
All colonies (transformants vector + promoter ligated with gvp) and o/n culture became red, so probably RFP is still in there... What might be the problem? The vector with promoter self-ligated due to uncomplete digestion (done 15 July 2009) leading to the following fragments:
- vector+RFP linear
- vector
- RFP
When the upper fragment (~2kb) is contaminated with linear vector this would lead high efficiency self-ligation instead of ligating gvp with the emtpy vector.
- →TODO Therefore the restriction/purification/ligation will be redone and selection of non-red colonies will be made!
- →DONE Also plasmid from the o/n cultures will be purified and analysed by restriction analysis.
- Used Sigma Plasmid isolation kit.
- DNA concentrations:
Transporters
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Metal Accumulation
Vectors
Dry
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