Melbourne/Lab BL Notebook/20070916PCR1
From 2007.igem.org
Protocol for PCR reactions A~G
For amplifying the photoreceptor and transmembrane domains of NpSopII-NpHtrII.
| PCR mix | PCR program | PrimerII |
|---|---|---|
| 5ul 10x buffer\\
5ul Enhancer 0.6ul dNTPs (25mM stock) 2.5ul MgSO4 (Supplied in PCR kit) 1.5ul Primer BL_FP1_s (10uM stock) 1.5ul Primer II (10uM stock) 1ul Template (pJS010) 0.4ul Pfx Platinum (Invitrogen) 32.5ul ddH2O | 94°C - 5'
94°C - 30" 59°C - 35" 68°C - 1.5' (goto step 2 x30) 68°C -10' 4°C forever | Reaction A => Primer BL_Con1_as (Some non-specific bands)
Reaction B => Primer BL_Con2_as Reaction C => Primer BL_Con3_as (Some non-specific bands) Reaction D => Primer BL_Con4_as Reaction E => Primer BL_Con5_as Reaction F => Primer BL_Con6_as Reaction G => Primer BL_Con7_as |
| 50ul Total |
Protocol for PCR reactions 1-7
For amplification of the kinase domain of ComP
| PCR mix | PCR program | PrimerI |
|---|---|---|
| 5ul 10x buffer\\
2.5ul MgSO4 (Supplied in PCR kit) 1.5ul Primer I (10uM stock) 1.5ul Primer VR (10uM stock) 1ul Template (pJS010) 0.4ul Pfx Platinum (Invitrogen) 32.5ul ddH2O | 94°C - 5'
94°C - 30" 59°C - 35" 68°C - 1.5' (goto step 2 x30) 68°C - 10' 4°C forever | Reaction A => Primer BL_Con1_s
Reaction B => Primer BL_Con2_s Reaction C => Primer BL_Con3_s Reaction D => Primer BL_Con4_s Reaction E => Primer BL_Con5_s Reaction F => Primer BL_Con6_s Reaction G => Primer BL_Con7_s |
| 50ul Total |
Gel Purification of PCR products A~G and 1~7
PCR product of the expected size was excised from .8% agarose gel and gel purified using the Invitrogen gel purification kit. Protocol as detailed in the kit.
