Difference between revisions of "Part:BBa K1539002:Design"

 
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===Design Notes===
 
===Design Notes===
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RBS primer construction needed to maintain the EcoRI and XbaI site. The promoter primer design could not maintain the EcoRI site but does contain the XbaI sequence. EcoRI was reestablished when the entire coding sequence was ligated to the backbone.  
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mCherry from BBa_J0650 was amplified with BBa_K1539034.  This product was Dpn1 digested and then PCR cleaned up. The product was then PCR re-amplified with BBa_K1539089, digested with XbaI and PstI and ligated into a similarly digested pSB1c3 backbone. Amplification of each step was confirmed by gel electrophresis producing bands approximately 900-950 bp (~700 bp for mCherry, 30 bp for RBS, 60 bp for promoter, and ~150 in extra sequence added by codons beyond the suffix to the reverse sequencing primer, VR).
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===Source===
 
===Source===

Latest revision as of 20:59, 1 November 2014


LPromoter->LRBS->mCherry


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

mCherry from BBa_J0650 was amplified with BBa_K1539034. This product was Dpn1 digested and then PCR cleaned up. The product was then PCR re-amplified with BBa_K1539089, digested with XbaI and PstI and ligated into a similarly digested pSB1c3 backbone. Amplification of each step was confirmed by gel electrophresis producing bands approximately 900-950 bp (~700 bp for mCherry, 30 bp for RBS, 60 bp for promoter, and ~150 in extra sequence added by codons beyond the suffix to the reverse sequencing primer, VR).

Source

mCherry from BBa_J06504 RBS and promoter from created primers

References