Jessica Karen Wong/Notebook/2007-7-2

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To Do

  • Retransform and plate RBS tester w/ RFP
  • Sequence promoter tester w/ RFP
  • Get part numbers for both
  • design BB primers
  • PCR T9002 and I2055

Registry

  • The RBS tester with RFP (that we used to call blue) is I2056
  • The promoter tester with RFP (used to call green) is I2057
  • Nishant sent I2057 for sequencing
  • To Recap: I2055 is the GFP RBS tester, E0240 is the GFP promoter tester, and T9002 is the output measurement tester (aka inverter, terminator, etc)

BioBrick Primers

  • E0240 needs a BioBrick site b/t the plasmid and the RBS
    • Forward Primer should match B0032 and have a Spe1 tail
    • Reverse Primers should have an EcoR1 tail and 1 should match 3K3 and the other 1AK3
  • I2055 needs a BioBrick site between the promoter and GFP
    • Fwd should match GFP (E0040) and have a Spe1 tail
    • Rev should match R0040 and have an EcoR1 tail
  • T9002 needs a BioBrick site between F2620 and B0032
    • Fwd should match B0032 and have Pst1 tail
    • Rev match Lux pR of F2620 and have EcoR1 tail

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

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

PCR

  • We did a 100ul preparatory PCR on T9002 at 54.5C
  • PCRed I2055 on a gradient 41-51
  • Diluted the new primers to 40uM
    • I2055 shorter F - 918 ul water
    • I2055 shorter R - 801 ul
    • I2055 longer R - 867.5 ul
    • T9002 R GFP - 801 ul