Team:Cambridge/Notebook/Week4

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Revision as of 11:31, 5 August 2010

Notebook: Week 4

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

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

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

GM230 hns-205 ::Tn10 TetR (black) - somewhat competent

W3110 hns 93-1 (blue) - no transformants

BW25113 Δ hns ::kan (red) - somewhat

W3110 hns-205::Tn10 Tet^R - no tansformants

Miniprep plasmid DNA from luciferase and backbone transformed TOP10 following Qiagen protocol
Digest luciferase Biobrick with: XbaI, PstI to produce two fragments:
1. P end---old backbone---E--Xend
2. X_end--luciferase---S--P_end (desired)
Digest backbone Biobrick with SpeI, PstI to produce:
1. S_end--short fragment--P_end
2. P_end---backbone--E--X--promoter--RBS--S_end (desired)
Run both digest products on a gel
Extract suitable lengthed fragments from gel:
1. Promoter + backbone should be 2153 bp
2. Luciferase should be 1653 bp (its 3426 bp backbone should be discarded)
Perform ligation
1. S and X are compatible so we get
  P_end---backbone--E--X--promoter--RBS--SX_scar--luciferase---S--P_end
2. The two P_ends will also ligate, producing a circular desired plasmid
Transform competent TOP10 cells with ligation product

@@ Line 29: / Line 29: @@
 ===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>
+W3110 hns 93-1 (blue) - no transformants
+BW25113 Δ  hns ::kan (red) - somewhat
+W3110 hns-205::Tn10 Tet<sup>R</sup> - no tansformants
 ===Theo's plan===