Team:Cambridge/Notebook/Week4
From 2010.igem.org
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==Thursday== | ==Thursday== | ||
+ | ===Results=== | ||
+ | GM230 hns-205 ::Tn10 TetR (black) - somewhat competent | ||
+ | W3110 hns 93-1 (blue) | ||
+ | BW25113 Δ hns ::kan (red) | ||
+ | W3119 hns-205::Tn10 Tet<sup>R</sup> | ||
+ | |||
+ | |||
===Theo's plan=== | ===Theo's plan=== | ||
# Miniprep plasmid DNA from luciferase and backbone transformed TOP10 following Qiagen protocol | # Miniprep plasmid DNA from luciferase and backbone transformed TOP10 following Qiagen protocol |
Revision as of 11:25, 5 August 2010
Notebook: Week 4
Monday
- Wet work
- Transformed competent cells using plasmid phK724 from Jim Slock (thanks!)
- Grew up E. coli containing phK555 in 5ml LB broth in rotating 37°C incubator overnight
- Dry work
- Finalising sequencing requirements
- Dismissed PPK
- Decided to include Luciola cruciata
- Further Wiki work
- Finalising sequencing requirements
Tuesday
- Wet Work
- Made competent cells of 4 different hns mutants: W3110 hns93-1, BW25113 Δhns::kan, W3110 hns-205::Tn10, GM230 hns-205::Tn10
- Now stored in the -80°C freezer for later use
- Dry Work
- Organising sequences for synthesis
Wednesday
- Hannah and Theo tested previous day's preparations for competence
- Peter and Paul transformed phK555 with phK724, placed cells in 30C incubator (above rotating incubator)
- Bill, Emily, Anja performed miniprep and restriction digest then ligation.
- Decided that we may have used wrong technique, but DNA samples in fridge
Thursday
Results
GM230 hns-205 ::Tn10 TetR (black) - somewhat competent W3110 hns 93-1 (blue) BW25113 Δ hns ::kan (red) W3119 hns-205::Tn10 TetR
Theo's plan
- Miniprep plasmid DNA from luciferase and backbone transformed TOP10 following Qiagen protocol
- Digest luciferase Biobrick with: XbaI, PstI to produce two fragments:
- P end---old backbone---E--Xend
- Xend--luciferase---S--Pend (desired)
- Digest backbone Biobrick with SpeI, PstI to produce:
- Send--short fragment--Pend
- Pend---backbone--E--X--promoter--RBS--Send (desired)
- Run both digest products on a gel
- Extract suitable lengthed fragments from gel:
- Promoter + backbone should be 2153 bp
- Luciferase should be 1653 bp (its 3426 bp backbone should be discarded)
- Perform ligation
- S and X are compatible so we get
Pend---backbone--E--X--promoter--RBS--SXscar--luciferase---S--Pend - The two Pends will also ligate, producing a circular desired plasmid
- S and X are compatible so we get
- Transform competent TOP10 cells with ligation product
Friday
Saturday
Sunday