Help:Standardization
Revision as of 15:52, 16 June 2006 by Smelissali (Talk | contribs) (→Removing the Biobricks Restriction sites on your sequence)
Contents
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
- First off, check your DNA sequence for the presence of 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://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
- Next, locate and remove all of the BioBrick standard restriction sites (XbaI, SpeI, EcoRI, PstI) that are on your sequence of interest. 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]
What you've got now: A cleaned-up area of sequence with no Biobricks restriction sites in it
Adding BioBrick Ends
- If your gene is small enough....Create primers with the Biobricks standard restriction sites according to Standard Assembly and PCR out the results
- If your gene is large...We recommend getting your gene synthesized, but be forewarned, this can be a costly process with issues about intellectual property (more on this later...)