Team:Cambridge/Experiments/Assembly of Reflectin Constructs

From 2011.igem.org

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==Assembly: first attempt==
==Assembly: first attempt==
===PCR===
===PCR===
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In the first round of PCR, we amplified fragments required for the assembly of GA1, GA2, GA3 and GA4 constructs.
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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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*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:
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*The time profile used in the PCR machine was the following:
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{| border="1" align="center" style="text-align:center;"
{| border="1" align="center" style="text-align:center;"
|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.
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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.
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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 12:00, 19 August 2011

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OVERVIEW
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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. 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?


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