Difference between revisions of "Help:Standardization"
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==Starting out== | ==Starting out== | ||
| − | '''What you want:''' A | + | '''What you want:''' A BiobBrick standardized sequence of interesting DNA |
''So I know of this interesting gene...'' | ''So I 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: | 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? | + | *What organism and strain is it in? Is it difficult to work with in the lab? |
*How well studied is the gene? | *How well studied is the gene? | ||
| − | *How long is the sequence? Is it so long ( | + | *How long is the sequence? Is it so long (>10 kb) that it might cause problems? |
| − | *What is the A-T:G-C content? If the ratio is skewed badly to one side, it can cause trouble with techniques like PCR assays (because of a very high annealing temperature) | + | *What is the A-T:G-C content? If the ratio is skewed badly to one side, it can cause trouble with techniques like PCR assays (because of a very high annealing temperature, for example). |
A good place to start looking for sequence is [http://www.ncbi.nlm.nih.gov/Genbank/index.html NCBI's Genbank] | 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:''' A non- | + | '''What you've got now:''' A non-BioBrick-standardized sequence of DNA that does something interesting |
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Revision as of 19:22, 29 May 2007
Contents
Starting out
What you want: A BiobBrick standardized sequence of interesting DNA
So I 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 organism and strain is it in? Is it difficult to work with in the lab?
- How well studied is the gene?
- How long is the sequence? Is it so long (>10 kb) that it might cause problems?
- What is the A-T:G-C content? If the ratio is skewed badly to one side, it can cause trouble with techniques like PCR assays (because of a very high annealing temperature, for example).
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: A 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 automatically done when you Add a part! Just enter the sequence of your part, and the Registry will give you the base pair coordinates of unwanted restriction sites in your part.
What you've got now: A piece of sequenced DNA with identified Biobricks restriction sites that you need to remove!
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, these are some popular procedures:
- 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], and there's more information in this [http://www.stratagene.com/manuals/200518.pdf Stratagene Mutagenesis Protocol (PDF download)]
- 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
Information, specifics, and sequence of the characterisic Biobrick Prefix and Suffix can be found here.
- If your gene is small enough... Order primers with the Biobricks end sequences (available in Part:BBa_G00000 and Part:BBa_G00001) as well as the aformentioned page on prefix and suffix sequence 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...)
What you've got now: Pieces of DNA with Biobrick ends in solution ready to be ligated and transformed into a containing plasmid vector
Links
- [http://parts.mit.edu/wiki/index.php/BU06:Research The BU iGEM team] is working on standardizing the Lux operon into a Biobrick using these techniques with a really spirited walkthrough and log of their progress