From OpenWetWare
Jump to navigationJump to search

Keeping track of construction files

You'll be doing lots of cloning--so much so that you probably won't remember how you made any particular constructs 6 months from now. It is therefore imperative that you keep some sort of log that shows what you've made, how you made it, and what it did. The Registry is essentially a database that stores information about Biobrick sequences. All the data eventually gets deposited in this Registry. Unfortunately, it's not so good right now as a design tool. New software tools for doing design are being developed, but until we have those, we have to do things based on more traditional means. Basically, you store all the information about construction and characterization in text files and spreadsheets, and then later move that data to the Registry. Here is a general description of how to store the construction information:

The text-style construction file

You've already been introduced to the text-based basic construction file in the tutorial which looked like this:

 Construction of KanR Basic Part Bca9128
 PCR ca1067F/R on pSB1AK3-b0015           (1055bp, EcoRI/SpeI/DpnI)
 Sub into pSB1A2-I13521                   (EcoRI/SpeI, 2062+946, L)
 Product is pSB1A2-Bca9128  [KanR]
 ca1067F  Forward Biobricking of KanR of pSB1AK3  ccagtGAATTCgtccTCTAGAgagctgatccttcaactc
 ca1067R  Reverse Biobricking of KanR of pSB1AK3  gcagtACTAGTtccgtcaagtcagcgtaatg

Always do something like this if you do anything fancy with your cloning like SOEing, restriction site removal, fill ins, blunting, etc. Basically, if it isn't just a PCR, cut, and paste, do this style. You will store these files in a page on the wiki.

The worksheet-style construction file

Whenever I order oligos, I create a spreadsheet file that includes the construction files in a slightly different format. Here is an example of one. You could keep these things on your desktop or as Google documents. You'll notice that our above construction file is encapsulated in the first line of the spreadsheet. So, it's much more compact, and is a nice guide to print out and put on your bench as you set up your PCRs if you want to do lots of cloning in parallel. Do these only if the construction file is really simple, ie, no removal of internal restriction sites or other PCR tricks.

The All-Oligos spreadsheet

The second spreadsheet document that goes along with this is the "all oligos" spreadsheet. Here is an example. You'll find it useful to have all the oligos in your freezer in one nice central location. When you want to find an oligo that can be used for sequencing, you can easily pairwise align the whole list to your sequence and see what hits. It also keeps all the information in one place so you don't have to hunt for files.

Biobrick calculator spreadsheets

You may find it useful to maintain a personal list of your Biobricks (and non-Biobrick sequences) in one file. Download this for an example. Surprisingly, both Google spreadsheets and excel can handle these files despite the large amount of data that accumulates -- mine is currently about 500 constructs long with no problems (wish me luck). You'll need to play with it to see how it works. Basically, you have basic parts and composite parts just like the Registry. You enter the part portion of the basic parts in a cell along with its name and description. Composite parts get calculated from the basic parts based on the user-supplied lefty and righty parts and the vector name. When you want to retrieve the complete sequence of a plasmid, you put the plasmid name and part name into the "plasmid calculator" and let it rip. You can then copy and paste the calculated sequence into ApE, hit <ctrl> K to annotate, and you have a nice annotated map to play with. Also, it doesn't care about the Biobrick standards or if there is a standard at all--it can handle your entire collection in the same document. You will use this type of data storage for all your composite parts. For all basic parts or non-Biobrick parts, you will use the text-based construction files or the worksheet-style construction file.