Going throuh our wiki

From 2007.igem.org

(Difference between revisions)
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==Brief description of how we used the wiki as a tool for molecular biology work==
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----
 
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Brief description of how we used the wiki as a tool for molecular biology work.
 
 +
Our wiki has proven to be an invaluable tool in organizing work within iGEM Paris 2007.
-
Our wiki has been proven to an invaluable tool in organising work within iGEM Paris 2007.
 
-
 
+
Molecular biology based engineering of DNA constructs involves going through a series of standardized steps, including:
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Molecular biology based engineering of desired constructs involves going through a series of standardized steps, including:
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*performing PCR reactions.
*performing PCR reactions.
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*Screening of E.coli colonies for correct construct
*Screening of E.coli colonies for correct construct
*Miniprep extraction of plasmids & subsequent sequencing of constructs
*Miniprep extraction of plasmids & subsequent sequencing of constructs
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Back to digestion step
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then going throuh the wholeprocess again.
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The nature of the work imposed by iGEM means that we had to go through this process several times, for a great number of different constructs, often being unsuccesfull and going through different steps several times.
+
The nature of the work imposed by iGEM means that we had to go through this process several times, for a great number of different constructs, often being unsuccessful and going through various steps several times.
-
We therefore organised the wiki in order to allow us to precisely keep track of our work:
+
We therefore organized the wiki in order to allow us to precisely keep track of our work:
Our [[Paris/Notebook Calendar|notebook]] is available on the wiki. It was mainly used for the wetlab experiments. A calendar allows following progress on a daily basis.
Our [[Paris/Notebook Calendar|notebook]] is available on the wiki. It was mainly used for the wetlab experiments. A calendar allows following progress on a daily basis.
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A sheet, describing the reaction, was filled and added to the notebook. The templates of the sheets are as follow:
A sheet, describing the reaction, was filled and added to the notebook. The templates of the sheets are as follow:
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PCR template:
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PCR sheet template:
{{Paris_PCR_0| Title =  
{{Paris_PCR_0| Title =  
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|}}
|}}
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Digestion template:
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All the primers purchased during the corse of the summer are listed in a special page and carry individual code numbers. See [[Paris/Oligos|primer collection]].
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Digestion sheet template:
{|
{|
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| style="background: #ccffcc;" |Digestion code nbre
| style="background: #ccffcc;" |Digestion code nbre
|
|
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|
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|ligation or PCR products
|
|
|
|
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|}
|}
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Ligation reactions:
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Ligation sheet reactions template:
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|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |ligation code nbre
| style="background: #ccffcc;" |ligation code nbre
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|
|
|
|
|
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All the primers purchased during the corse of the summer are listed in a special page and carry individual code numbers. See [[Paris/Oligos|primer collection]].
+
In addition, each time one of these reactions was successful, the corresponding sheet line was copies into our database on the [[Paris/Freezer|FREEZER]] page of the wiki.  
-
 
+
In order to further enhance traceability, each time a unique digestion, ligation or PCR reaction was performed, it was given an individual code number: respectively Dx, Lx & Px.  
-
In addition, each time one of these reactions was successful, the corresponding sheet line was copies into the database on the [[Paris/Freezer|freezer]] page of the wiki.
+
A given reaction product can thus be identified by: its code number & the date at which the reaction was performed.  
-
 
+
-
In order to further facilitate travability, each time a unique digestion, ligation or PCR reaction was performed, it was given an individual code number: respectively Dx, Lx & Px.  
+
-
A given reaction product can thus be identified by: its code & the date at which the reaction was performed.  
+
If a given reaction (for instance: digestion product Dx: digestion of plamid x by EcoRI & SpeI enzymes and extraction of the small fragment which can serve as a Forward insert) was performed several time, the reaction product would keep the same code number. These different products would be distinguished by the date.
If a given reaction (for instance: digestion product Dx: digestion of plamid x by EcoRI & SpeI enzymes and extraction of the small fragment which can serve as a Forward insert) was performed several time, the reaction product would keep the same code number. These different products would be distinguished by the date.
We tried to do this for most of our work and we believe we have achieved a reasonable degree of traceability.
We tried to do this for most of our work and we believe we have achieved a reasonable degree of traceability.
 +
 +
 +
==Historie of reaction steps of the DNA construction process==
 +
 +
In addition, we have included a graph showing the different reaction steps leading to the main constructs we generated. There is no need for you to go through this process now, as each construction history graph is added whenever a construct of interest is introduced in the description.
 +
 +
 +
 +
=== Upstream construct ===
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<html>
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<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=6,0,29,0" width="800" height="600">
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<param name="movie" value="http://poupitoupou.free.fr/Construct_FW.swf">
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<param name="quality" value="high">
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<embed src="http://poupitoupou.free.fr/Construct_FW.swf" quality="high" pluginspage="http://www.macromedia.com/go/getflashplayer" type="application/x-shockwave-flash" width="500" height="750"></embed></object>
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</html>
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=== Downstream construct ===
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==== Intermediate construct ====
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<br>
 +
<html>
 +
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=6,0,29,0" width="800" height="600">
 +
<param name="movie" value="http://poupitoupou.free.fr/Construct_Intermediate.swf">
 +
<param name="quality" value="high">
 +
<embed src="http://poupitoupou.free.fr/Construct_Intermediate.swf" quality="high" pluginspage="http://www.macromedia.com/go/getflashplayer" type="application/x-shockwave-flash" width="400" height="500"></embed></object>
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</html>
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====  Backward construct with DapA E.Coli ====
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<br>
 +
<html>
 +
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=6,0,29,0" width="800" height="600">
 +
<param name="movie" value="http://poupitoupou.free.fr/Construct_BW_DapE.swf">
 +
<param name="quality" value="high">
 +
<embed src="http://poupitoupou.free.fr/Construct_BW_DapE.swf" quality="high" pluginspage="http://www.macromedia.com/go/getflashplayer" type="application/x-shockwave-flash" width="450" height="350"></embed></object>
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</html>
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====  Backward construct with DapA Subtilis ====
 +
<br>
 +
<html>
 +
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=6,0,29,0" width="800" height="600">
 +
<param name="movie" value="http://poupitoupou.free.fr/Construct_BW_DapS.swf">
 +
<param name="quality" value="high">
 +
<embed src="http://poupitoupou.free.fr/Construct_BW_DapS.swf" quality="high" pluginspage="http://www.macromedia.com/go/getflashplayer" type="application/x-shockwave-flash" width="600" height="550"></embed></object>
 +
</html>
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 +
=== Recombinaison rate measurement ===
 +
<br>
 +
<html>
 +
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=6,0,29,0" width="800" height="600">
 +
<param name="movie" value="http://poupitoupou.free.fr/Construct_MRR.swf">
 +
<param name="quality" value="high">
 +
<embed src="http://poupitoupou.free.fr/Construct_MRR.swf" quality="high" pluginspage="http://www.macromedia.com/go/getflashplayer" type="application/x-shockwave-flash" width="500" height="600"></embed></object>
 +
</html>

Revision as of 19:22, 26 October 2007

Contents

Brief description of how we used the wiki as a tool for molecular biology work

Our wiki has proven to be an invaluable tool in organizing work within iGEM Paris 2007.


Molecular biology based engineering of DNA constructs involves going through a series of standardized steps, including:

  • performing PCR reactions.
  • Digesting DNA.
  • Ligating digested DNA products.
  • Transforming competent E.coli cells with ligation reaction products.
  • Screening of E.coli colonies for correct construct
  • Miniprep extraction of plasmids & subsequent sequencing of constructs

then going throuh the wholeprocess again.

The nature of the work imposed by iGEM means that we had to go through this process several times, for a great number of different constructs, often being unsuccessful and going through various steps several times.

We therefore organized the wiki in order to allow us to precisely keep track of our work:

Our notebook is available on the wiki. It was mainly used for the wetlab experiments. A calendar allows following progress on a daily basis.


Each time any one of the following reactions was performed

  • DNA digestion reaction
  • PCR reaction
  • Ligation reaction

A sheet, describing the reaction, was filled and added to the notebook. The templates of the sheets are as follow:

PCR sheet template:

PCR :
PCR Settings Buffer (5x) µl x Expected size
Annealing (°C) MgCl2 10µM [] {{{Size}}}
° dNTP 10µM [] Success
Time Elongation Oligo F 10µM (number of the Fw primer) µL {{{Success}}}
‘ Oligo R 10µM (number of the Rv primer) µL Image (click to enlarge)
Number cycles Water µL [[Image:{{{Image}}}|30px]]
x Polymerase Band (0=ladder)
DNA {{{Band}}}

All the primers purchased during the corse of the summer are listed in a special page and carry individual code numbers. See primer collection.


Digestion sheet template:

Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 Size Description
Digestion code nbre ligation or PCR products

Ligation sheet reactions template:


Ligations
Number Insert Insert Volume (µL) Vector Vector Volume (µL) Comments Number of colonies
ligation code nbre


In addition, each time one of these reactions was successful, the corresponding sheet line was copies into our database on the FREEZER page of the wiki.

In order to further enhance traceability, each time a unique digestion, ligation or PCR reaction was performed, it was given an individual code number: respectively Dx, Lx & Px. A given reaction product can thus be identified by: its code number & the date at which the reaction was performed. If a given reaction (for instance: digestion product Dx: digestion of plamid x by EcoRI & SpeI enzymes and extraction of the small fragment which can serve as a Forward insert) was performed several time, the reaction product would keep the same code number. These different products would be distinguished by the date.


We tried to do this for most of our work and we believe we have achieved a reasonable degree of traceability.


Historie of reaction steps of the DNA construction process

In addition, we have included a graph showing the different reaction steps leading to the main constructs we generated. There is no need for you to go through this process now, as each construction history graph is added whenever a construct of interest is introduced in the description.


Upstream construct

Downstream construct

Intermediate construct


Backward construct with DapA E.Coli


Backward construct with DapA Subtilis


Recombinaison rate measurement


Retrieved from "http://2007.igem.org/wiki/index.php/Going_throuh_our_wiki"