User:Mackenzie L. Cowell/IGEM 2007 part fab protocol
The 2007 DNA part kit will be comprised of around 1500 parts contained on 4 384-well plates. We need to fabricate 150 copies of the kit, producing a total of 600 384-well plates. The parts are contained on plasmids in cultures of E. coli frozen in glycerol stocks in 96-deep-well (96d) plates in -80 freezers at the registry. We are going to inoculate 96d plates from the freezer stocks and give them to Ed's lab for incubation and then miniprepping in 4 groups of 4, for a total of 16 96d plates. For every 4 that we get back we will use the EPM to compose the miniprepped DNA into 9 384-well intermediate source plates (SP), of which we will give 5 back to Ed's lab to use with the PMP to transfer 3.0 uL of DNA into 150 384-well kit plates. This will happen 4 times, producing an uncollated set of the 600 384-well plates for the 150 07 DNA part kits.
The 07 kits will be comprised of no more than 4 384-well plates (1536 parts), so to make 150 kits, we'll need:
- 32 96-deep-well square-bottom plates
- 16 for Ed's miniprep, will be sources for the intermediate SPs
- 16 for miniprepping for Robotic Assembly supplies
- 16 96-deep-well round-bottom plates (for 2 glycerol stocks of each new registry freezer plate)
- 20 384-deep-well plates
- 16 for Antibiotic Testing
- 4 for Robotic Assembly supplies
- 660 384-well plates, for kits (4 per kit)
- 600 for kits (4 per kit)
- 60 for intermediate Source Plates for kit fab with the PMP (the PMP 384 tip head can't reach the bottom of 384d plates)
- 672 foil covers
656 plastic covers(AB test plates don't need plastic covers & the plates come with them already)
- 15 rolls label tape
- 32 boxes of tips
(Items that are crossed out have been delivered to the Registry and are stored in 314)
96d miniprep cultures
Making a fresh culture of one of the 96d freezer (library) plates involves filling each well of the new plate with LB and the particular antibiotics used to select for the part clone in the corresponding well of the freezer plate. Randy can generate a .csv file that instructs the EPM to add the antibiotics for any given freezer plate (antibiotic resistance can also be read manually from the Physical DNA section of the part's entry in the registry).
8 miniprep culture plates can be prepared in one EPM run (2 miniprep cultures need to be made for each freezer plate), leaving room on the EPM stage for two boxes of tips and the reservoir of antibiotics. The volume of antibiotic needed in one run is equal to: 8 plates * 96 wells/plate = 768 wells, * 20 uL / well = 15,360 uL
The EPM will add 20 uL of the correct antibiotic to each well, resulting in a 1:10 dilution. If the antibiotic stocks are normally diluted 1:1000 to obtain a working concentration, then they should be diluted 1:100 when put into the EPM reservoir (not necessarily true for tet).
- Prepare 150 mL of LB-amp (768 wells * 180 uL/ well = 138.240 mL)
- Pre-fill each well of the 96d plates with with 180 uL of LB-amp (amp conc: 100 ug/mL)
- Use the EPM to do this with method file notlisted.ext, or
- Use the plate-filler in the deLong lab to do it
- Fill reservoir 1 with 20 mL chloramphenicol at 350 ug/mL
- Fill reservoir 2 with 20 mL kanamycin at 500 ug/mL
- Fill reservoir 3 with 20 mL tetracycline at 150 ug/mL
- howto: import .csv file(s)?
- howto: Make sure each plate is in the correct position on the stage
- list: settings in EPM method file: what tip head to use, how long the run will take, etc.
- Run the EPM
- when finished, replace the covers on each culture plate and store in the refrigerator.
Library plates 1-8 exist in triplicate and are spread across the two Knight lab and one of the Endy lab -80 freezers for redundancy. Duplicate glycerol stocks of the newer, singular library plates (9-16) contributing to the 2007 parts kits should thus be made when the library plates are thawed to inoculate the miniprep plates.
Like the miniprep plates, each well of the 96d glycerol stock plates needs the particular combination of antibiotics compatible with the part clone that will be grown and stored there, and thus the production process for making the glycerol stocks is similar to that for making the miniprep plates.
Based on the glycerol stock protocol in the Quigen bench guide:
- Each well should hold logarithmic growth cultures mixed to be 15% Glycerol by volume.
384d antibiotic test
Antibiotics (also see BU protocol)
Typical concentrations and volumes
|ampicillin||100 mg/mL||100 ug/mL|
|kanamycin||50 mg/mL||50 ug/mL|
|chloramphenicol||25 mg/mL||25 ug/mL|
|tetracycline||5 mg/mL||15 ug/mL|
|plate||well volume||total volume||manufacturer||spec sheet|
|96d||2 mL||192 ml||?||?|
|384||110 uL||42.240 mL||nunc||242747|
|384d||220 uL||84.480 mL||?||?|
Test Run: 10 April 07
Meet Sultrim around noon with 6 384 plates filled with 100 uL dyed dH20 per well (230.4 mL ) and 20 empty 384 well plates. Try filling the empty plates with 3.0 uL from the SPs.