Difference between revisions of "Help:Standardization"
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<b>What you've got:</b> Any non-Biobrick-standardized sequence of DNA that does something interesting<br>
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==Starting out==
 
<b>What you want:</b>A Biobrick standardized sequence of interesting DNA
 
<b>What you want:</b>A Biobrick standardized sequence of interesting DNA
 +
 +
''So I've know of this interesting gene...''<br>
 +
Find that sequence!  This can be a lot harder than it sounds...but make sure you can find out the following specifics:
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*What strain is it in?  Is it difficult to work with in the lab?
 +
*How well studied is the gene?  <br>
 +
A good place to start looking for sequence is [http://www.ncbi.nlm.nih.gov/Genbank/index.html NCBI's Genbank]
 +
 +
<b>What you've got now:</b> Any non-Biobrick-standardized sequence of DNA that does something interesting<br>
  
 
<hr>
 
<hr>
 
So here's how you do it...
 
So here's how you do it...
  
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#First off, check your DNA sequence for restriction sites.  This is easy using available web programs such as
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==Checking for Restriction sites==
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*First off, check your DNA sequence for the presence of [https://parts.igem.org/cgi/htdocs/Assembly/restriction_enzymes.cgi Biobricks restriction sites].  This is easy using available web programs such as
 
#*[http://tools.neb.com/NEBcutter2/index.php NEB cutter] - by New England Biolabs
 
#*[http://tools.neb.com/NEBcutter2/index.php NEB cutter] - by New England Biolabs
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#*[http://wishart.biology.ualberta.ca/PlasMapper/ PlasMapper] - developed by the University of Alberta
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#*[http://wishart.biology.ualberta.ca/PlasMapper/ PlasMapper] - developed by the University of Alberta<br>
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#Next, locate and remove all of the BioBrick standard restriction sites (XbaI, SpeI, EcoRI, PstI) that are on your sequence.  To remove these sites, you there are a few processes based on the same concept as PCR amplification that you can use:
+
 
 +
<b>What you've got now:</b>A piece of sequenced DNA with identified Biobricks restriction sites that you need to get rid of!
 +
 
 +
==Removing the Biobricks Restriction sites on your sequence==
 +
#Next, locate and remove all of the BioBrick standard restriction sites (XbaI, SpeI, EcoRI, PstI) that are on your sequence.  To remove these sites, you there are a few processes based on the same concept as PCR amplification. Here are some protocols that you can use:
 
#*''site directed mutagenesis'' - best for changing small, targeted areas of the genome like the Biobricks standard restriction sites.  Primers with the desired mutation align to the area you would like to change, and then PCR amplification extends them.  Openwetware has a nice [http://openwetware.org/wiki/Site-directed_mutagenesis protocol on how to do this].
 
#*''site directed mutagenesis'' - best for changing small, targeted areas of the genome like the Biobricks standard restriction sites.  Primers with the desired mutation align to the area you would like to change, and then PCR amplification extends them.  Openwetware has a nice [http://openwetware.org/wiki/Site-directed_mutagenesis protocol on how to do this].
 
#*''lambda red'' - best for if you have very large (thousands of kb, entire genes) areas for alteration or deletion.  This process is laid out in [http://www.pnas.org/cgi/content/full/97/12/6640 Datsenko and Wanner, 2000]<br>  
 
#*''lambda red'' - best for if you have very large (thousands of kb, entire genes) areas for alteration or deletion.  This process is laid out in [http://www.pnas.org/cgi/content/full/97/12/6640 Datsenko and Wanner, 2000]<br>  
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<small>''Note: When designing your primers, keep in mind where the open reading frame (ORF) is, and remember that you might need to substitute a codon for a similar amino acid<small>''
 
 
#Add the Biobricks standard restriction sites according to [https://parts.igem.org/cgi/htdocs/Assembly/index.cgi Standard Assembly]
 
#Add the Biobricks standard restriction sites according to [https://parts.igem.org/cgi/htdocs/Assembly/index.cgi Standard Assembly]
 +
#*
 +
<hr>

Revision as of 15:48, 16 June 2006

Starting out

What you want:A Biobrick standardized sequence of interesting DNA

So I've know of this interesting gene...
Find that sequence! This can be a lot harder than it sounds...but make sure you can find out the following specifics:

  • What strain is it in? Is it difficult to work with in the lab?
  • How well studied is the gene?

A good place to start looking for sequence is [http://www.ncbi.nlm.nih.gov/Genbank/index.html NCBI's Genbank]

What you've got now: Any non-Biobrick-standardized sequence of DNA that does something interesting

So here's how you do it...

Checking for Restriction sites

    • [http://tools.neb.com/NEBcutter2/index.php NEB cutter] - by New England Biolabs
    • [http://wishart.biology.ualberta.ca/PlasMapper/ PlasMapper] - developed by the University of Alberta

What you've got now:A piece of sequenced DNA with identified Biobricks restriction sites that you need to get rid of!

Removing the Biobricks Restriction sites on your sequence

  1. Next, locate and remove all of the BioBrick standard restriction sites (XbaI, SpeI, EcoRI, PstI) that are on your sequence. To remove these sites, you there are a few processes based on the same concept as PCR amplification. Here are some protocols that you can use:
    • site directed mutagenesis - best for changing small, targeted areas of the genome like the Biobricks standard restriction sites. Primers with the desired mutation align to the area you would like to change, and then PCR amplification extends them. Openwetware has a nice [http://openwetware.org/wiki/Site-directed_mutagenesis protocol on how to do this].
    • lambda red - best for if you have very large (thousands of kb, entire genes) areas for alteration or deletion. This process is laid out in [http://www.pnas.org/cgi/content/full/97/12/6640 Datsenko and Wanner, 2000]
  2. Add the Biobricks standard restriction sites according to Standard Assembly