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Team:Cambridge/Experiments/Assembly of Reflectin Constructs
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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 eventually we did not detect any products on the gels. | *Primers and template DNA provided by our supervisor Paul served as a positive control, but eventually we did not detect any products on the gels. | ||
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The pictures below present result of gel electrophoresis of PCR products. | The pictures below present result of gel electrophoresis of PCR products. | ||
Revision as of 11:43, 25 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 eventually we did not detect any products on the gels.
The pictures below present result of gel electrophoresis of PCR products.
- In most cases position of a band matches the expected length of DNA fragment. The only exception are GA1-2 and GA3-2 products. According to the position on the gel the length of these DNA fragments is 4-5kb, whereas the predicted length is 3.5kb. Our hypothesis is that we were provided [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] backbone instead of [http://partsregistry.org/Part:pSB1A3 pSB1A3]backbone.
- For GA1-1, GA2-1, GA3-1 and GA4-1 we obtained two bands -
- The molecular weight marker that we used in all gels is HyperLadder I, which produces regularly spaced bands ranging from 200 to 10,000bp.
Gibson Assembly
Transformation
Results
Diagnostics
Assembly: second attempt
PCR
Gibson Assembly
Transformation
Results
What next?
