Team:Cambridge/Notebook/Week4

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{{:Team:Cambridge/Templates/headerbar|colour=#fad72a|title=Notebook: Week 4}}
 
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==Monday==
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{{:Team:Cambridge/Templates/HybridBook|num=4}}
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*Wet work
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**Transformed competent cells using plasmid phK724 from Jim Slock (thanks!)
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**Grew up E. coli containing phK555 in 5ml LB broth in rotating 37°C incubator overnight
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*Dry work
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**Finalising sequencing requirements
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***Dismissed PPK
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***Decided to include Luciola cruciata
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**Further Wiki work
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==Tuesday==
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* Wet Work
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** Made competent cells of 4 different hns mutants: W3110 hns93-1, BW25113 Δhns::kan, W3110 hns-205::Tn10, GM230 hns-205::Tn10
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** Now stored in the -80°C freezer for later use
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* Dry Work
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** Organising sequences for synthesis
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==Wednesdy==
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==Thursday==
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===Theo's plan===
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# Miniprep plasmid DNA from luciferase and backbone transformed TOP10 following Qiagen protocol
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# Digest luciferase Biobrick with: XbaI, PstI to produce two fragments:
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## P end---old backbone---E--Xend
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## Xend--luciferase---S--Pend <strong>(desired)</strong>
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# Digest backbone Biobrick with SpeI, PstI to produce:
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## Send--short fragment--Pend
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## Pend---backbone--E--X--promoter--RBS--Send <strong>(desired)</strong>
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# Run both digest products on a gel
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# Extract suitable lengthed fragments from gel:
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## Backbone should be 2153 bp
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## Luciferase should be 1653 bp (its 3426 bp backbone should be discarded)
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# Perform ligation
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##P and X are compatible so: we get Pend---backbone--E--X--promoter--RBS--SXscar--luciferase---S--Pend
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##The two P ends will also ligate, producing a circular desired plasmid
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# Transform competent TOP10 cells with ligation product
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==Friday==
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==Saturday==
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==Sunday==
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<html>
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</div>
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</html>
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{{:Team:Cambridge/Templates/footerMinimal}}
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Latest revision as of 22:43, 18 August 2010