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

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 * style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]] Magnetic Bacteria
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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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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


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