User:Karmella Haynes/Notebook/BioBrick cloning/2010/08/05

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  • ✓ Sequencing order: KAH160-170
  • ✓ Order oligos: seq primers for pAct5C/ KFV1
  • ✓ Transformation (quick and dirty): pCaSpeR-hs/eGFP-Nintra plasmid (from J. Belote) into T-DH5α

Sequencing order
> Confirm that Kozak is at the beginning of constructs
--> old vector from insert digest could yield false positive
--> Kozak too short to tell difference between old insert vs. new construct
--> Forward: P0001

  1. KAH160-1 F: good (keep)
  2. KAH160-2 F: good
  3. KAH161-1 F: good (keep)
  4. KAH161-2 F: good
  5. KAH162-1 F: good (keep)
  6. KAH162-2 F: good
  7. KAH163-1 F: good (keep)
  8. KAH163-2 F: good
  9. KAH164-1 F: good (keep)
  10. KAH164-2 F: bad read (non-specific)
  11. KAH165-1 F: okay, seq match after manual corrections (keep)
  12. KAH165-2 F: bad read (non-specific)
  13. KAH166-1 F: fshPcCD, should be flyPcCD; good seq (keep)
  14. KAH166-2 F: same
  15. KAH167-1 F: flyPcCD, should be fshPcCD; good seq (keep)
  16. KAH167-2 F: same
  17. KAH168-1 F: good (keep)
  18. KAH168-2 F: bad read (poor quality)
  19. KAH169-1 F: bad read (poor quality); keep and use based on digest result from 8/04/10
  20. KAH169-2 F: bad read (no priming)
  21. KAH170-1 F: good (keep)
  22. KAH170-2 F: good

Order oligos

> Ian (Depace lab) sequenced off the end of the R. luciferase gene in pAct5C using 5'-ATCGGACCCAGGATTCTTTT. Use this sequence data to design reverse sequencing primers for reading back towards the R. luc insert (the spot where I cloned in the microMCS).


> "Nintra" fly deg. tag BB primers and sd mutagenesis primer
--> See Neuburger et al. 2006, Genetics for details on eGFP-Nintra

  1. BB_Nintra f; 5'-cctttctagaAAGAATAGTGCAATAATGCAAACG
  2. BB_Nintra r; 5'-aaggctgcagcggccgctactagtAATGTAGATGGCCTCGGAAC