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Team:Cambridge/Experiments/Assembly of Reflectin Constructs
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===PCR=== | ===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. | 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: | The time profile used in the PCR machine was the following: | ||
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Revision as of 12:01, 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 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?
