Team:Cambridge/Experiments/Assembly of Reflectin Constructs

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(→Primer Design)
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In the first round of PCR, we amplified fragments required for the assembly of GA1, GA2, GA3 and GA4 constructs.
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.
*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:
 +
{|
 +
|Hold
 +
|95
 +
|2min
 +
|-
 +
|Cycling
 +
|Denaturing
 +
|95
 +
|10s
 +
|-
 +
|
 +
|Annealing
 +
|55
 +
|20s
 +
|-
 +
|
 +
|Elongation
 +
|72
 +
|150s
 +
|}
 +
 +
*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.
===Gibson Assembly===
===Gibson Assembly===

Revision as of 11:34, 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.

Hold 95 2min
Cycling Denaturing 95 10s
Annealing 55 20s
Elongation 72 150s
  • 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.

Gibson Assembly

Transformation

Results

Diagnostics

Assembly: second attempt

PCR

Gibson Assembly

Transformation

Results

What next?


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