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

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)