From 2011.igem.org
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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.
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| - | *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="1px" align="center" style="text-align:center;"
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| - | |Hold
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| - | |95
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| - | |2min
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| - | |-
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| - | |Cycling
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| - | |Denaturing
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| - | |95
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| - | |10s
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| - | |-
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| - | |
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| - | |Annealing
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| - | |55
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| - | |20s
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| - | |-
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| - | |
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| - | |Elongation
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| - | |72
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| - | |150s
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| - | |}
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| - | *We decided to use 55 degrees annealing temperaure, although the calculated temperature for most primers is 5-10 degrees higher, because of low annealing temperature of the VF2 primer.
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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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| - | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}
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Latest revision as of 14:46, 15 September 2011
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