Plasmid Preparation Machine

			Highlights See panels from G.S.A.C. '98 presentation

Preparing plasmid DNA templates involves harvesting bacterial cells before lysing and removing all genomic DNA, bacterial proteins and any salts that might be present. State of the art miniprep kits currently cost >$1/well and require close to an hour of technician time per plate. 

We have designed a robot that will handle approx. 6 96-well bacterial culture plates per hour without human intervention. At the same time we have reduced the cost of the prep to <$0.10/well. This robot is currently in construction. 

			96 well plate

The robot utilizes a patented 96-well flow-through microcentrifuge developed in our group. Cell pelleting and resuspension have traditionally been performed manually in a 96 well plate centrifuge and vortexer. The automation of these steps allows plates of bacterial cultures to be processed directly without technician intervention. Resuspension is achieved by counter-rotation of the centrifuge rotors which maximizes shear flow across the pellet. Mixing of samples within the rotor can be automated in a similar way which allows all necessary protocol steps to be completed without moving the sample out of the rotor. A 96 channel device prototype is currently being tested. The footprint of this device will be comparable to a 96 well microtiter plate allowing the robot to be accomodated into a small space. 

			rotor

We are evaluating 3 protocols for use with our robot. All protocols use the flow-through microcentrifuge to generate cleared cell lysate from bacterial cultures via alkaline lysis. This step is then followed by: A) an ethanol precipitation performed in a second flow-through microcentrifuge, B) a purification by binding the DNA to glass filter fibers, or C) binding the DNA to silicate particles which are separated from solution in a second flow-through microcentrifuge. All of these have been tested in single channel format and have given good sequencing results (> 600 bp reads on ABI 377 using TaqFS dye primer reactions). The cost of these protocols range from $0.02/well for the ethanol ppt prep. to $0.12/well for the glass filter fiber prep. 

Protocol:

Buffers:

• Resuspension Buffer (P1):
  50 mM Tris-HCl
  10 mM EDTA
  pH 8.0
  ** Note no RNase required for glass filter fiber purification***
• Lysis Buffer (P2):
  .2 M NaOH
  1% SDS (Sodium Dodecylsulfate)
• Neutralization Buffer (P3):
  3 M KAc, pH 5.5
• Binding Buffer (B1):
  7M Guanidine-HCL in Water
• Wash Buffer (W1):
  70% Ethanol - 30% Water
• Elution buffer (E1):
  1 X TE

Additional Materials:

• Glass fiber filter:
  800 µl filter plate from Polyfiltronics
  GF-B + 5.0 um Polypropylene for support

Lysate Clearing Procedure:

1. Start with 300 µl of saturated bacterial culture. Typically cells grown in TB for 24 hours to saturation in pressurized oxygen atmosphere. O.D. at 600nm ~18.
2. Harvest cells in 96 well flow-through microcentrifuge. Discard supernatant.
3. Resuspend cells in 100 µl of P1 by counter-rotation of centrifuge rotors.
4. Add 100 µl of P2 and mix gently.
5. Incubate for 5 min.
6. Add 100 µl of P3 and mix gently.
7. Pellet cell debris and K/SDS precipitate by spinning up rotors to 20,000g.
8. Collect the cleared lysate supernatant.

A) Ethanol Precipitation Procedure:

1. Add 300 µl 100% Ethanol to collected lysate and load into flow-through microcentrifuge.
2. Spin rotors up to 20,000g for 3 min. Discard supernatant.
3. Fill rotors with W1.
4. Spin rotors up to 20,000g for 1 min. to retain pellet. Discard supernatant.
5. Dry pellet with compressed air.
6. Add 60 µl of E1 and spin rotors up to moderate speed to dissolve pellet.
7. Collect purified template.

B) Glass Filter Procedure:

1. Add 300 µl of B1 to collected lysate and load into glass filter plate.
2. Incubate for 2 min.
3. Run lysate through filter using positive pressure above the plate.
4. Load filter with 600 µl of W1.
5. Run wash buffer through filter using positive pressure above plate.
6. Repeat wash steps twice more.
7. Flow compressed air through the filter in order to dry off any remaining ethanol.
8. Add 60 µl of E1.
9. Incubate for 2 min.
10. Elute from the filter under high pressure.

Typical yields are 50 µl containing 100 ng/µl of plasmid DNA.