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 either the EPM or the PMP to compose the miniprepped DNA into 9 384-well intermediate source plates (SP), of which we will use 5 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.
- Prepare 8 96d miniprep plates
- just use 2mL LB-amp per well (1536 mL total LB-amp)
- Prepare 16 AB plates (one for each unique library plate)
- Thaw library plates 1-4 (96d glycerol stocks)
- Inoculate the miniprep and AB plates,
- if the pen-tool is available
- use it to inoculate 2 miniprep plates for each library plate
- use 2 runs of the PMP to inoculate the AB plates
- use 2 runs of the PMP to inoculate the miniprep & AB plates
- return the library plates to the freezer
- cover miniprep plates with breathable tape
- cover the AB plates with foil
- if the pen-tool is available
- O/n incubate the AB plates and 4 miniprep plates at the registry
- Deliver the other 4 inoculated 96d miniprep plates to Ed's lab for o/n incubation and miniprepping. Also supply 4 new 96d plates for the miniprepped DNA to be eluted into. Request an elution volume of 1000 uL.
- Check AB test plate and update registry
- Process 4 miniprep plates incubated at registry for Robotic assembly intermediates
- Retrieve 4 miniprepped plates from Ed's lab
- use EPM to add 10 uL of dye into each well of the 4 MP plates
- use EPM to compose 180 uL from each miniprep plate into 6 96d plates (4 EPM runs); label these source plates (SP) 1234.1-1234.6
- store the SP in the refrigerator ?
- take 3 SP and 150 384 kit-plates and 150 labels over to the PMP.
- use the pmp to transfer 3 uL from each SP into 50 kit plates, leaving a residual volume of DNA in the SP of 30 uL. Remove plates in groups of 10 from the stackers and visually inspect all wells for missing DNA. If it's ok, cover each plate with its plastic lid and label the bottom. Program the PMP to pause after filling 50 plates so the SP can be replaced and to prevent it from getting too far ahead of the visual inspection (it can transfer the 3 uL from the SP to the kit plate in about 35 seconds).
- bring the SPs back to the lab for drying
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 1:100 dilution. If the antibiotic stocks are normally diluted 1:1000 to obtain a working concentration, then they should be diluted 1:10 when put into the EPM reservoir (not necessarily true for tet: 14ml LB + 6 tet stock in reservoir).
- Prepare 150 mL of LB-amp (768 wells * 180 uL/ well = 138.240 mL)
- Pre-fill each well of the 96d plates with with 1980 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.
Before inoculation, the well of a glycerol plate should consist of 792 uL LB-amp (100 ug/mL amp) 8 uL specific antibiotic.
384d antibiotic test
All part clones will be tested for correct antibiotic resistance during the inoculation of the miniprep plates by also inoculating a 384d antibiotic test (AB) plate. The wells of the AB plates are grouped into four quadrants such that each well of a 96d library plate corresponds to 4 contiguous wells on the AB plate, with each of the 4 correspondent wells containing a different antibiotic broth.
We can prepare all the AB plates in advance with the EPM.
- Use 4 methods, one for each antibiotic.
- 100ul EZ Rich broth with appropriate antibiotic in each well.
100ul * 96 wells = 9600ul of each antibiotic broth per plate * 8 plates per run = 76,800ul of broth per run (20ml per reservoir * 4 reservoirs)
Typical concentrations and volumes
Antibiotics (also see BU protocol)
|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.