User:Mackenzie L. Cowell/IGEM 2007 part fab protocol

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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.


Necessary Supplies

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)

Preparing Supplies

96d miniprep cultures

96d glycerols

384d antibiotic test

Antibiotics (also see BU protocol

Typical concentrations and volumes

antibiotic stock working
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
96  ?  ?  ?  ?
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.