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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==Wednesday==
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*Hannah and Theo tested previous day's preparations for competence
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*Peter and Paul transformed phK555 with phK724, placed cells in 30C incubator (above rotating incubator)
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*Bill, Emily, Anja performed miniprep and restriction digest then ligation.
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**Decided that we may have used wrong technique, but DNA samples in fridge
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==Thursday==
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===Results===
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GM230 hns-205 ::Tn10 TetR (black) - somewhat competent
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W3110 hns 93-1 (blue) - no transformants
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BW25113 Δ  hns ::kan (red) - somewhat
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W3110 hns-205::Tn10 Tet<sup>R</sup> - no tansformants
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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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## X<sub>end</sub>--luciferase---S--P<sub>end</sub> <strong>(desired)</strong>
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# Digest backbone Biobrick with SpeI, PstI to produce:
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## S<sub>end</sub>--short fragment--P<sub>end</sub>
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## P<sub>end</sub>---backbone--E--X--promoter--RBS--S<sub>end</sub> <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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## Promoter + 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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##S and X are compatible so we get <br />P<sub>end</sub>---backbone--E--X--promoter--RBS--SX<sub>scar</sub>--luciferase---S--P<sub>end</sub>
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##The two P<sub>end</sub>s 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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Latest revision as of 22:43, 18 August 2010