From 2011.igem.org
(Difference between revisions)
|
|
| (163 intermediate revisions not shown) |
| Line 1: |
Line 1: |
| - | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}
| |
| | | | |
| - | This is a placeholder. We should fill it in.
| |
| - |
| |
| - | ==Construct Design==
| |
| - | ==Primer Design==
| |
| - | We should mention expected lengths of products here.
| |
| - |
| |
| - | ==Assembly: first attempt==
| |
| - | ===PCR===
| |
| - | 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.
| |
| - | *The time profile used in the PCR machine was the following:
| |
| - | {| border="1px" align="center" style="text-align:center;"
| |
| - | |Hold
| |
| - | |95
| |
| - | |2min
| |
| - | |-
| |
| - | |Cycling
| |
| - | |Denaturing
| |
| - | |95
| |
| - | |10s
| |
| - | |-
| |
| - | |
| |
| - | |Annealing
| |
| - | |55
| |
| - | |20s
| |
| - | |-
| |
| - | |
| |
| - | |Elongation
| |
| - | |72
| |
| - | |150s
| |
| - | |}
| |
| - |
| |
| - | *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.
| |
| - |
| |
| - | ===Gibson Assembly===
| |
| - | ===Transformation===
| |
| - | ===Results===
| |
| - | ===Diagnostics===
| |
| - | ==Assembly: second attempt==
| |
| - | ===PCR===
| |
| - | ===Gibson Assembly===
| |
| - | ===Transformation===
| |
| - | ===Results===
| |
| - | ==What next?==
| |
| - |
| |
| - | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}
| |
Latest revision as of 14:46, 15 September 2011
"
