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| - | {{:Team:BCCS-Bristol/Header}}
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| - | {{:Team:BCCS-Bristol/NotebookHeader}}
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| - | ===Week 8===
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| - | * pQE31-GFP was digested and ligations with digested and purified FhuA were performed.
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| - | * XL1-Blue competent cells were transformed with the pQE31-GFP+FhuA ligations.
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| - | * The concentration of the DNA parts that we are going to send for sequencing was determined by spectrophotometry. These parts include: AraC-RBS, AraC-RBS-FhuA/OsmE, GFP-terminator and bioscaffolds-GFP-terminator.
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| - | * Liquid cultures of the bioscaffolds-GFP-terminator were prepared and minipreps performed to isolated the plasmid DNA. Then the whole bioscaffold-GFP-terminator constructs were purified by gel extraction and ligations with the AraC-RBS-FhuA/OsmE were prepared.
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| - | * Overnight ligations set-up;
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| - | 1) AraC-RBS-FhuA-Bioscaffold-GFP-Terminator
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| - | 2) AraC-RBS-OsmE-Bioscaffold-GFP-Terminator
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| - | * Prepare liquid cultures for pQE31-GFP-FhuA.
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| - | =====Promoter-RBS-GFP-terminator ligation=====
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| - | * This will be used to check that GFP works and also to check if the AraC promoter works by trying to control its level of activity using arabinose.
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| - | * Restriction digest and gel purify GFP-terminator part and then ligate it to the already cut AraC-RBS on pSB1A2 plasmid.
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| - | [[Image:BCCS_Arac_rbs_gfp_term.png|center|500px|frame|The figure illustrates the construct AraC-RBS-GFP-TErminator created to assess promoter activity. The ladder is 1kb DNA marker from NEB. Lanes run in triplets with Double Digest (XbaI/Osme), Single Digest(XbaI) and Uncut versions of DNA plasmids carrying the construct. Presence is illustrated by the bands created in the double digest lane (top:plasmid backbone bottom:construct)]]
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Latest revision as of 23:48, 20 October 2009
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