IGEM:Cambridge/2008/Notebook/Magnetic Bacteria/2008/07/30

From OpenWetWare

Jump to: navigation, search


Magnetic Bacteria Main project page
Previous entry      Next entry

Change of Plan...!

Change of Plan after Discussion with Nice Helpful Postgrads...!:

  • Directly ligate gene (mms6/mamC/magA) onto plasmid pSB2K3 carrying promoter I0500 and terminators
  • Incubate 8μL TOP10 competent cells in 10ml LB at 37°C for 3 hours
  • Transform TOP10 with I0500 plasmid and MagA plasmid
  • I0500: 1μL of biobrick DNA + 50μL TOP10
  • MagA: 2μL of DNA + 50μL of TOP10
  • Followed standard transformation protocol for TOP10

PCR MagA

  • PCR MagA gene with MagA primers received from sigma
  • Notice different annealing temperature for the forward and reverse primers
    • 85.4°C for MagAF and 79.4°C for MagAR
  • 100μM stock of primers prepared from dry primers
    • Add 210μL SDW for MagAF and 417μL SDW for MagAR
  • 10μM stock prepared by 10μL of 100μM in 100μL SDW
  • New PCR Programme in E-gel PCR Machine (> Shared > MagA)
  • primer temp = 79°C and 34 cycles
  • 10μL of MagA PCR product kept in freezer
  • other 10μL of MagA PCR product with 3μL dye and 7μL SDW into well of E-gel (1.2% SyBr Green)

Result:

  • PCR did not work as we need to incorporate overhang when considering the PCR programme and the primer temperature.
  • Temperature should be:
    • MagAF = 50°C
    • MagAR = 49°C
  • Preffix and suffix might not have annealed at the higher temperature we've used



Personal tools