Registry/Measurement kit/Notebook/2007-7-2

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Key

  • I2055 - GFP RBS tester
  • I2056 - RFP RBS tester (blue)
  • I2057 - RFP promoter tester (green)

Retransformation of I2057

  • Used previous ligation (tet and chlor)
  • Plated 200uL

Sequence check of I2057

  • dnalims.mit.edu
  • Mix for sequencing rxn
    • 1uL DNA (200-500 ng)
    • 2uL 1.6 uM VF2/VR
    • 9uL H2O

Scarring PCR

  • Gradient PCR of I2055 from 41 to 51 deg
  • PCR of T9002 with Tm of 54.5 deg

Primer Design

To insert BB sites. Format: 8-mer.restriction site.primer

  • E0240:
    • F (incl E0240, PstI): CTTAGTAG.ACTAGT.TCACACAGGAAAGTACTAGATGCG (53.1)
    • R (incl 3K3/1AK3, EcoRI): TCAGCGAT.GAATTC.CAGAAATCATCCTTAGCGAAAGC (54.2)
  • I2057:
    • F (incl I2057, PstI): CTTAGTAG.ACTAGT.TCACACAGGAAAGTACTAGATGGC (52.1)
    • R: same as E0240
  • I2055:
    • F (incl GFP, SpeI): CTTAGTAG.ACTAGT.ATGCGTAAAGGAGAAGAACTTTTC (52.4)
    • R (incl R0040, EcoRI): TCAGCGAT.GAATTC.GTGCTCAGTATCTCTATCACTGATAGG (52.0)
  • I2056:
    • F (incl RFP, SpeI): CTTAGTAG.ACTAGT.ATGGCTTCCTCCGAAGACG (54.0)
    • R: Same as I2055
  • For T9002:
    • F (incl lux pR, PstI): TCAGCGAT.CTGCAG.TCACACAGGAAAGTACTAGATGCG (53.1)
    • R (incl RBS, GFP,EcoRI) : CTTAGTAG.GAATTC.TTTATTCGACTATAACAAACCATTTTC (51.9)