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Project Documentation & Protocols: Maize Gene Discovery Project: RescueMu Library Plates: Protocols: Library Plate Production

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Plasmid Rescue

RescueMu Protocol

  1. Thaw frozen E.coli libraries on ice if not from freshly prepared cells.
  2. Plate adequate amount (300 Ál) of each row/column library on a LB+Carb (100 mg/L) plate evenly with a sterile spreader.
  3. Invert the plates and let them grow overnight at 37C.
  4. Make one set of replica plates. Let both the original and the replica sets of plates grow further at 37C for 4-6 hours and overnight, respectively.
  5. If the colonies on the original set of plates are not fully grown (~1 mm), let them sit on the bench at room temperature overnight. Those fully grown can be wrapped with parafilm straps and stored at 4C. Save the original set for sequencing and other purpose.
  6. To each replica plate, add 7~8 ml of LB+Carb (100 mg/L) medium. Resuspend the colonies with a sterile spreader and collect into four 2-ml tubes.
  7. Centrifuge for 2 min at maximum speed in a microcentrifuge. Discard the supernatant and store the bacterial pellet at .20C until ready for plasmid extraction.
  8. Medium-throughput plasmid miniprep using Qiaprep-8 with a vacuum manifold.
  9. Dispense 200 ng / 10 Ál of each row/column library in a well of a deep 96-well plate.

Plate format:
Table containing details on the library plate format

Note: Whenever possible, multiple plates and/or rescues of a library may be necessary to obtain a reasonable number of colonies. For grid G (46x48 at 100% transposition), the average colony counts per library was ~200 with the minimum ~100.


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