IGEM:University of Chicago/2009/Notebook/Paraoxon Biosensor/2009/06/24

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June 24, 2009

  • Picked up plate of W3031A +ADE2 S. cerevisae. Put in fridge. Lots of nice coloneis
  • Plated 2 plates (NORA and ROB) of SKB4182 at 11:30am. Will come in later tonight to make 3mL cultures for mini-preps
SKB4182 is Longtine plasmid pFA6a-GFP-KanMX. Used for homologous recombination in S. cerevisiae, W3031A.
  • Need to order primers for homologous recombination, using Longtine primers F2 and R1, and DNA overlaps to Schofield genes (use top 7).

Primers

  • Ordered primers for Longtine constructs and Schofield genes from Invitrogen (standard desalting)

Deletion constructs

Construction of YGR035C deletion, replacement by GFP via homologous gene recombination in S. cerevisiae

PCR YGR035Cdel-F/YGR035Cdel-R on PCR YGR035CFor/YGR035CRev on pFA6a-GFP(S65T)-kanMX6           (2504bp, EcoRI/SpeI/DpnI)
Sub into YGR035C gene on Chr. VII                   
Product is delYGR035C-GFP  [KanR]

  • YGR035Cdel-F: 63-mer CGGAAGTTATAAGTTATCTTCTCTACAGAAACAATACGTAATGcggatccccgggttaattaa
  • YGR035Cdel-R: 62-mer GAAGATGCAAAGAAGTAGTTAAGCACATTGACCTCAGGGTTGgaattcgagctcgtttaaac


Construction of YHR139C deleProxy-Connection: keep-alive Cache-Control: max-age=0

on, replacement with GFP via homologous gene recombination in S. cerevisiae

PCR YHR139Cdel-F/YHR139Cdel-R on pFA6a-GFP(S65T)-kanMX6           (2504bp, EcoRI/SpeI/DpnI)
Sub into YHR139C gene on Chr. VIII                   
Product is delYHR139C-GFP  [KanR]

  • YHR139Cdel-F: 65-mer GATTTCGATCAGTTTTCACATCTAAGAACCCAATCAGCTATTATGcggatccccgggttaattaa
  • YHR139Cdel-R: 60-mer ATTTTTTGCTTATTTTCGTAGTACACAACACATAATCATCgaattcgagctcgtttaaac


Construction of YOR049C deletion, replacement with GFP via homologous gene recombination in S. cerevisiae

PCR YOR049Cdel-F/YOR049del-R on pFA6a-GFP(S65T)-kanMX6           (2504bp, EcoRI/SpeI/DpnI)
Sub into YOR049C gene on Chr. XV               
Product is delYOR039-GFP [KanR]

  • YOR049Cdel-F: 60-mer GAAGCAGCAGCTAACAGTAAATTCCCTTTATGGTGGTATGcggatccccgggttaattaa
  • YOR049Cdel-R: 60-mer CATATGAACGATCAGATTGACATTCAAATTCTACGTATCAgaattcgagctcgtttaaac


Construction of YOR186W deletion, replacement with GFP via homologous gene recombination in S. cerevisiae

PCR YOR186WCdel-F/YOR186Wdel-R on pFA6a-GFP(S65T)-kanMX6           (2504bp, EcoRI/SpeI/DpnI)
Sub into YOR186W gene on Chr. XV               
Product is delYOR186W-GFP [KanR]

  • YOR186WCdel-F: 64-mer GTTTACTCCTTCTCTCAAATACTACGTAACAGATCTTATTGATGcggatccccgggttaattaa
  • YOR186Wdel-R: 66-mer GTTTTAAATGAGAAAAGCAGCTTCTTTTCCTTTCTTCCACGTAGTAgaattcgagctcgtttaaac


Construction of YGR213C deletion, replacement with GFP via homologous gene recombination in S. cerevisiae

PCR YGR213Cdel-F/YGR213Cdel-R on pFA6a-GFP(S65T)-kanMX6           (2504bp, EcoRI/SpeI/DpnI)
Sub into YGR213C gene on Chr. VII              
Product is delYGR213C-GFP [KanR]

  • YGR213Cdel-F: 62-mer GTATGTAATAGACAGTCAGCACTTTTCACCACTTCGAACATGcggatccccgggttaattaa
  • YGR213Cdel-R: 61-mer GAATAAAACCACATTTCCCACATGTAAATATAGAAAGCAGTgaattcgagctcgtttaaac

Construction of YGR236C deletion, replacement with GFP via homologous gene recombination in S. cerevisiae

PCR YGR236Cdel-F/YGR236Cdel-R on pFA6a-GFP(S65T)-kanMX6           (2504bp, EcoRI/SpeI/DpnI)
Sub into YGR236C gene on Chr. VII              
Product is delYGR236C-GFP [KanR]

  • YGR236Cdel-F: 63-mer CAAATATTTAGCGCATAAAATTCAAACAAAGTTTACTGAAATGcggatccccgggttaattaa
  • YGR236Cdel-R: 65-mer CAAAATGCAAAGAACATAAATGCAGGGAACCAAGTACAAATTCCCgaattcgagctcgtttaaac


Construction of YLR346C deletion, replacement with GFP via homologous gene recombination in S. cerevisiae

PCR YLR346Cdel-F/YLR346del-R on pFA6a-GFP(S65T)-kanMX6           (2504bp, EcoRI/SpeI/DpnI)
Sub into YLR346C gene on Chr. XII                  
Product is delYLR346C-GFP [KanR]

  • YLR346Cdel-F: 62-mer GGGGCACAAATAACAAAGGATTATTTATCACCTTTAATAATGcggatccccgggttaattaa
  • YLR346Cdel-R: 62-mer GATATGAGGGGACGATAGCTTAACATGATTGTGACACAAACTgaattcgagctcgtttaaac




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