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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Magnetic Bacteria
