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| - | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}
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| - | This is a placeholder. We should fill it in.
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| - | ==Construct Design==
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| - | ==Primer Design==
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| - | We should mention expected lengths of products here.
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| - | ==Assembly: first attempt==
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| - | ===PCR===
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| - | In the first round of PCR, we amplified fragments required for the assembly of GA1, GA2, GA3 and GA4 constructs. We performed PCR using [[Team:Cambridge/Protocols/PCR#Reagents_used_in_PCR_reaction | Phusion Hot Start DNA Polymerase]] in 20 μl reaction volume.
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| - | The time profile used in the PCR machine was the following:
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| - | {| border="1" align="center" style="text-align:center;"
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| - | |scope="col" width="50" | '''Hold'''
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| - | |95°C
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| - | |2 min
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| - | |-
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| - | |scope="col" width="50" rowspan="3" | '''Cycling'''
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| - | |scope="col" width="80" | ''Denaturing''
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| - | |scope="col" width="50" | 95°C
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| - | |scope="col" width="50" | 10 s
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| - | |-
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| - | |scope="col" width="80" | ''Annealing''
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| - | |scope="col" width="50" | 55°C
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| - | |scope="col" width="50" | 20 s
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| - | |-
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| - | |scope="col" width="80" | ''Elongation''
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| - | |scope="col" width="50" | 72°C
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| - | |scope="col" width="50" | 150 s
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| - | |}
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| - | We decided to use the 55°C annealing temperaure, although the predicted temperature for most primers is 5-10°C higher, because of a low annealing temperature of the VF2 primer.
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| - | Primers and template DNA provided by our supervisor Paul served as a positive control, but we did not detect any products on a gel.
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| - |
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| - | ===Gibson Assembly===
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| - | ===Transformation===
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| - | ===Results===
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| - | ===Diagnostics===
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| - | ==Assembly: second attempt==
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| - | ===PCR===
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| - | ===Gibson Assembly===
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| - | ===Transformation===
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| - | ===Results===
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| - | ==What next?==
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| - |
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| - | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}
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