Team:Cambridge/Experiments/Assembly of Reflectin Constructs

From 2011.igem.org

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*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.
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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.
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*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 11:57, 19 August 2011

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OVERVIEW
home

This is a placeholder. We should fill it in.

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.

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?


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