Melbourne/Lab BL Notebook/20070916PCR1
From 2007.igem.org
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4°C forever | 4°C forever | ||
| - | |*<Reaction A1, A, 1> | + | | |
| + | *<Reaction A1, A, 1> | ||
*<Reaction B2, B, 2> | *<Reaction B2, B, 2> | ||
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*<Reaction C3, C, 3> | *<Reaction C3, C, 3> | ||
| - | <Reaction D4, D, 4> | + | *<Reaction D4, D, 4> |
| - | <Reaction E5, E, 5> | + | *<Reaction E5, E, 5> |
| - | <Reaction F6, F, 6> | + | *<Reaction F6, F, 6> |
| - | <Reaction G7, G, 7> | + | *<Reaction G7, G, 7> |
|- | |- | ||
| 50ul Total | | 50ul Total | ||
|} | |} | ||
Revision as of 15:30, 10 October 2007
Contents |
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 10x 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 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 1 => Primer BL_Con1_s
Reaction 2 => Primer BL_Con2_s Reaction 3 => Primer BL_Con3_s Reaction 4 => Primer BL_Con4_s Reaction 5 => Primer BL_Con5_s Reaction 6 => Primer BL_Con6_s Reaction 7 => 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.
Second Round PCR
For stitching products A~G to 1~7. Gel purified PCR products from the above reaction were used.
| PCR mix | PCR program | <Reaction, TemplateI, TemplateII> |
|---|---|---|
| 5ul 10x buffer\\
2.5ul 10x Enhancer 0.6ul dNTPs (25mM stock) 2.5ul MgSO4 (Supplied in PCR kit) 1.5ul Primer BL_FP1_s (10uM stock) 1.5ul Primer VR (10uM stock) 5ul Template I {A~G} 5ul Template II {1-7} 0.4ul Pfx 27ul ddH2O | 94°C - 5'
94°C - 1' 50°C - 1' 68°C - 3' (goto step 2 x30) 68°C - 10' 4°C forever |
|
| 50ul Total |
