Team:Groningen/Notebook/17 July 2009

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Revision as of 10:24, 17 July 2009

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

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

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

Metal Accumulation

Vectors

Dry