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Team:Cambridge/Experiments/Assembly of Reflectin Constructs
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==Assembly: first attempt== | ==Assembly: first attempt== | ||
===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. | |
| - | The time profile used in the PCR machine was the following: | + | *The time profile used in the PCR machine was the following: |
{| border="1" align="center" style="text-align:center;" | {| border="1" align="center" style="text-align:center;" | ||
|scope="col" width="50" | '''Hold''' | |scope="col" width="50" | '''Hold''' | ||
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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. | 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. | |
| - | 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=== | ===Gibson Assembly=== | ||
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?
