Team:Cambridge/Notebook/Week4

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

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

Theo's plan

1. Miniprep plasmid DNA from luciferase and backbone transformed TOP10 following Qiagen protocol
2. 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)
3. 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)
4. Run both digest products on a gel
5. 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)
6. Perform ligation
   1. P 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
7. Transform competent TOP10 cells with ligation product

Friday

Saturday

Sunday