Difference between revisions of "Part:BBa K2765030:Design"

 
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===Design Notes===
 
===Design Notes===
  
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cloned the upstream homologous arm and the downstream homologous arm of yno1 from the genome of CEN.PK2-1C and cloned Leu marker from pESC-Leu.ligased Leu marker and the complete fragment by OE-PCR then transformed them into CEN.PK2-1C(△yca1).
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We designed 3 pairs of primers whose templates were the upstream homologous arm, the marker and the downstream homologous arm respectively. The marker was Uracil synthesis gene abbreviated as ura.Initially, we got 3 fragments by PCR and they had their own overlapping areas with corresponding to each homologous arms.Then, we got the recombination fragment from OE-PCR that has been transformed to the CEN.PK2-1C which has been already knocked out the endogenous genes ura. Additionally, the colony was chosen by the ura nutritional deficiency medium.
  
  

Revision as of 07:29, 17 October 2018


1000bp Homologous arm+Ura(△yca1)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2416
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1825
    Illegal BsaI.rc site found at 277
    Illegal SapI.rc site found at 559
    Illegal SapI.rc site found at 580
    Illegal SapI.rc site found at 1672


Design Notes

We designed 3 pairs of primers whose templates were the upstream homologous arm, the marker and the downstream homologous arm respectively. The marker was Uracil synthesis gene abbreviated as ura.Initially, we got 3 fragments by PCR and they had their own overlapping areas with corresponding to each homologous arms.Then, we got the recombination fragment from OE-PCR that has been transformed to the CEN.PK2-1C which has been already knocked out the endogenous genes ura. Additionally, the colony was chosen by the ura nutritional deficiency medium.



Source

S. cerevisiae CEN.PK2-1C


References