Difference between revisions of "Help:Standardization"
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#*[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 | ||
#*[http://wishart.biology.ualberta.ca/PlasMapper/ PlasMapper] - developed by the University of Alberta | #*[http://wishart.biology.ualberta.ca/PlasMapper/ PlasMapper] - developed by the University of Alberta | ||
| − | #Next, locate and remove all of the BioBrick standard restriction sites (XbaI, SpeI, EcoRI, PstI) that are on your sequence. To | + | #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: |
| − | #*site directed mutagenesis - best for changing | + | #*''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] | + | #*''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> |
| + | <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] | ||
Revision as of 15:28, 16 June 2006
What you've got: Any non-Biobrick-standardized sequence of DNA that does something interesting
What you want:A Biobrick standardized sequence of interesting DNA
So here's how you do it...
- First off, check your DNA sequence for 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://wishart.biology.ualberta.ca/PlasMapper/ PlasMapper] - developed by the University of Alberta
- 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:
- 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]
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
- Add the Biobricks standard restriction sites according to Standard Assembly