User:Karmella Haynes/Notebook/BioBrick cloning/2010/07/16

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07/16/10

  • ✓ Sequencing order: KHF01-04
  • ✓ Oligo order: micro BioBrick cloning site for pAct5C plasmid; micro BB cloning site for TetO-FUW-oct4 lentivirus vector



Sequencing order
> fwd = P0001, rev = P0002

  1. KHF01 fwd: good
  2. KHF01 rev: good
  3. KHF02-1 fwd: good
  4. KHF02-1 rev: good
  5. KHF02-2 fwd: n/d
  6. KHF02-2 rev: n/d
  7. KHF03-1 fwd: good
  8. KHF03-1 rev: good
  9. KHF03-2 fwd: n/d
  10. KHF03-2 rev: n/d
  11. KHF04-1 fwd: mutation at 575 bp (V > A missense mutation)
  12. KHF04-1 rev: mutation at 575 bp (V > A missense mutation)
  13. KHF04-2 fwd: good
  14. KHF04-2 rev: good



Oligo order

> micro MCS for pAct5C
--> "micro MCS site for BioBrick inclusion" from V0200: CACCgcggccgc(NotI)AGTGTCAAGGGTactagt(SpeI)C
--> Vector: Act5C promoter - SpeI - R. luciferase - NotI
--> Replace SpeI/NotI fragment with NotI/SpeI micro MCS; destroy SpeI w/ XabI overhang; destroy NotI site with a mutation before NotI overhang
--> Oligos:

  1. microMCS 1; 5'-ctagaCACCgcggccgcAGTGTCAAGGGTactagtC
  2. microMCS 2; 5'-ggccGactagtACCCTTGACACTgcggccgcGGTG


> micro MCS for TetO-FUW-oct4 lentivirus vector (for Pc-ATF expression in stem cells)
--> Replace EcoRI fragment with EcoRI/XbaI micro MCS; destroy second EcoRI site with a mutation before the EcoRI overhang
--> Oligos:

  1. microMCS2 1; 5'-aattcGAGGCCGCAtctagaTt
  2. microMCS2 2; 5'-aattaAtctagaTGCGGCCTCg