Team:Cambridge/Experiments/Assembly of Reflectin Constructs

From 2011.igem.org

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*The time profile used in the PCR machine was the following:
*The time profile used in the PCR machine was the following:
{| border="1px" align="center" style="text-align:center;"
{| border="1px" align="center" style="text-align:center;"
-
|scope="col" width="100" | '''Hold'''
+
|scope="col" width="50" | '''Hold'''
|
|
-
|95
+
|95°C
-
|2min
+
|2 min
|-
|-
-
|scope="col" width="80" | '''Cycling'''
+
|scope="col" width="50" | '''Cycling'''
|scope="col" width="80" | ''Denaturing''
|scope="col" width="80" | ''Denaturing''
-
|scope="col" width="50" | 95
+
|scope="col" width="50" | 95°C
-
|scope="col" width="50" | 10s
+
|scope="col" width="50" | 10 s
|-
|-
|
|
|scope="col" width="80" | ''Annealing''
|scope="col" width="80" | ''Annealing''
-
|scope="col" width="50" | 55
+
|scope="col" width="50" | 55°C
-
|scope="col" width="50" | 20s
+
|scope="col" width="50" | 20 s
|-
|-
|
|
|scope="col" width="80" | ''Elongation''
|scope="col" width="80" | ''Elongation''
-
|scope="col" width="50" | 72
+
|scope="col" width="50" | 72°C
-
|scope="col" width="50" | 150s
+
|scope="col" width="50" | 150 s
|}
|}

Revision as of 11:48, 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 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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