IGEM:Cambridge/2008/Notebook/Magnetic Bacteria/2008/07/25: Difference between revisions
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* Biobrick BBa_1006 of about 250bp used as reference | * Biobrick BBa_1006 of about 250bp used as reference | ||
* DNA run on 0.8% agarose gel with SyBr Green | * DNA run on 0.8% agarose gel with SyBr Green | ||
PCR protocol with 34 cycles: | PCR protocol with 34 cycles: | ||
* 5μL DNA | * 5μL DNA | ||
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* 10μL Finnzyme Master Mix | * 10μL Finnzyme Master Mix | ||
* Total volume of 20μL | * Total volume of 20μL | ||
[[Image:25jul08.jpg|frame|right|Gel showing I0500 PCR Product at about 1.2kb]] | |||
Protocol for preparation of SyBr Green 0.8% Agarose Gel: | Protocol for preparation of SyBr Green 0.8% Agarose Gel: | ||
* 0.8g of Ultrapure agarose powder | * 0.8g of Ultrapure agarose powder |
Revision as of 04:08, 7 August 2008
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Preparation for Plasmid AssemblyGenes mms6 and mamC
Protocol of Plasmid Restriction Digestion:
Plasmid Vector pSB3K3http://partsregistry.org/Part:pSB3K3
Promoter I0500http://partsregistry.org/Part:BBa_I0500
PCR protocol with 34 cycles:
Protocol for preparation of SyBr Green 0.8% Agarose Gel:
<html><br> Lane 3 - Hyperladder I<br> Lane 4 - I746:101<br> Lane 5 - I746:001<br> Lane 7 - I0500 promoter PCR product<br> Lane 8 - I006 control<br> <br></br></html> Result:
Preparation of M9 Medium (Iron Free)M9 salt and glucose solution is reduced in iron content using Chelex-100 resins, which are removed by filtration. Millipore filter used to pass the prepared M9 medium (glucose added) to sterilise the medium at the end. Sterilised deionised water should be used in the preparation of iron citrate stock. Preparation of M9 Medium 5X M9 salts contain:
For the M9 medium, Component Concentrations:
For 500ml of media:
Protocol:
Preparation of 200ml iron citrate stock (iron:citrate ratio = 1:100)Reagents needed: 1. Tri-sodium citrate
2. Ferrous sulfate FeSO4•7H2O
3. Aqueous sodium hydroxide
Ferrous sulfate should be added when needed in the experiment to prevent oxidation of Fe(II) to insoluble Fe(III) precipitates. Anaerobic condition needed when incubating E.coli to prevent such oxidation reaction which affects soluble iron ion concentration in solution and the iron uptake by bacteria.
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