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Team:Cambridge/Experiments/Assembly of Reflectin Constructs
From 2011.igem.org
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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 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.
- Primers and template DNA provided by our supervisor Paul served as a positive control, but we did not detect any products on a gel.
Gibson Assembly
Transformation
Results
Diagnostics
Assembly: second attempt
PCR
Gibson Assembly
Transformation
Results
What next?
