Revision as of 15:53, 1 May 2008

iGEM 2008 DNA Parts Kit

Background on 08 distribution

Quality Control Process

This year the Registry has undertaken extensive quality control measures in order to better ensure the accuracy of DNA parts sent to the iGEM 2008 teams. This QC process began in February and has examined each part currently in the Registry to determine if the plasmid and the part within are correct according to the documentation provided with each submission. The results for each part were then posted on the Registry's DNA repository page [ ], allowing iGEM teams to access this information before using the submissions to create their own parts.

Grouping of parts for QC project

Each part in the Registry was assigned a well in a specific 96 well staging plate according to its antibiotic resistance and its base pair length. This grouping made it easier to examine the parts against their known antibiotic resistance and to then sequence their DNA.

Streaking for Single Colonies

Once it was decided which part would be assigned to each plate, bacterial stocks containing parts were plated and streaked for single colonies.

Parts in the Registry prior to iGEM 2007 were kept in glycerol stocks in the Registry's -80C freezer, where they were directly streaked onto Petri dishes with appropriate antibiotic and grown overnight at 37C.

The rest of the parts - ones submitted by iGEM 2007 teams - were received as DNA and transformed into competent cells. Once transformed, they were also plated onto Petri dishes with appropriate antibiotic and allowed to incubate overnight at 37C.

Preparation of and Inoculation from Staging Plate

The next morning, Petri plates were removed from the incubator and a single colony was picked from each plate. Using a pipette tip, the colony chosen was put into a corresponding well of a 96 deep well plate filled with the appropriate antibiotic broth, creating a staging plate for those grouped bioparts.

Once all 96 wells of the staging plate have been inoculated with single colonies, a 5.0ul pin tool replicator was used to then inoculate from the staging plate to antibiotic testing plates,miniprep plates, and glycerol plates, and the plates were grown for 10hr - 14hr.

Four antibiotic testing plates were prepared, testing known bacterial resistance of ampicillin, kanamycin, chloramphenicol, and tetracycline. The plates were grown overnight at 37C, pelleted and examined for resistant growth in wells.

As well as the antibiotic testing plates, five glycerol plates containing 10% glycerol broth and appropriate antibiotic were prepared for the long term storage of the bacterial stocks. One of these plates will remain in the Registry -80C freezer while the others will be stored offsite.

Lastly, two miniprep plates were inoculated, incubated overnight and minipreped the following day. The DNA extracted from the cultures was resuspended in TE and 15.0ul of each sample digested and run on an electrophoresis get to examine the size of plasmid and part. In addition, 24.0ul was sent to the Broad Institute for DNA sequencing. The remaining DNA left from the miniprep was then spotted to grided filter paper to generate the spring 2008 DNA distribution.

Instruction on usage