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Team:Cambridge/Experiments/Assembly of Reflectin Constructs
From 2011.igem.org
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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. | *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: | *The time profile used in the PCR machine was the following: | ||
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|scope="col" width="50" | '''Hold''' | |scope="col" width="50" | '''Hold''' | ||
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Revision as of 11:48, 19 August 2011
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Contents |
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 Phusion Hot Start DNA Polymerase in 20 μl reaction volume.
- The time profile used in the PCR machine was the following:
| Hold | 95°C | 2 min | |
| Cycling | Denaturing | 95°C | 10 s |
| Annealing | 55°C | 20 s | |
| Elongation | 72°C | 150 s |
- 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?
