Millipore Plasmid Prep (centrifuge method)

Growing cultures:

1.  Use the deep-well blocks that come with the kit.

2.  Add 1ml/well 2xYT media with appropriate antibiotic (100ug/ml Amp).

3.  Pick colonies directly into wells or add appropriate amount of freezer stock

     (usually 10ul).

4. Cover the block only with the plastic lid that comes with it.

5. Grow overnight at 37°C shaking at 225rpm.

Pellet:

1.  Spin at 1500 x g for 5 minutes.

2.  Turn block upside down to dump media (into appropriate container) and tap on paper

      towels to remove excess liquid.  You can leave the blocks upside down on towels for

      a few minutes as long as the pellets aren’t coming loose.

3.  At this point, the blocks can be stored at -20°C or prepped.

Plasmid Prep:

1.  Resuspend the pellets by adding 150µl of Solution I to each well.

2.  Cover the block with a thermoseal plate sealer before vortexing.  The kit provides one

     sealer per block, but I used that one at the end for storing my samples.

3.  Vortex well to make sure the pellets are fully resuspended.

4.  Add 150µl of Solution 2 to each well.

5.  Vortex well for 1 minute.

6.  Let the block sit at room temperature for 2 minutes.  (**Do NOT exceed 5 minutes)

7.  Add 150µl of Solution 3 to each well.

8.  Vortex for 2 min.

9.  Assemble the Clearing Plate on top of the Plasmid Plate.  (I used one of the plastic

     plate rims that we use when putting a Millipore Filter plate on top of a non-skirted

     PCR plate in order to get each of the filter plates to sit firmly on top of each other.)

    Tape them together.  Then add a disposable V-bottom plate (reusable) to the bottom of

    the Plasmid Plate, and tape those together.

Clearing Plate

Plasmid Plate

Disposable V-bottom

10.  Add 300µl of sample from the deep-well block onto the Clearing Plate.  The rest of

       the sample and the block may be thrown away.

11.  Centrifuge at 1500rpm for 5 minutes.

12.  Remove the top Clearing Plate and discard.

13.  Also remove the V-bottom plate, dump the liquid, and reassemble to the bottom

       of the Plasmid Plate.

14.  Continue centrifuging at 1500rpm for 5 minutes.  Then dump V-bottom plate again.

15.  Add 200ul of Solution 4 to each well.

16.  Centrifuge at 1500rpm for 8 minutes.

17.  Remove the V-bottom plate, dump the waste, and set the plate aside.

18.  Recover the DNA from the Plasmid Plate by adding 50ul of Solution 5 to each well.

      **The wells appear shiny even when there is no liquid in them.

19.  Place the plate on a shaker for 5 min.  I used our loud plate shaker with the speed dial

       set at 3.  I think you could probably just vortex it a bit instead.  The protocol with the

       kit says you can alternatively leave it sitting on a bench top at room temperature for

       30 minutes.

20.  Pipette out all of the liquid (I had around 60µl instead of just the 50µl added at the           

       end) and transfer to a V-bottom plate (comes with the kit) for storage.  The kit also

       provides a plate sealer to use.

Sequencing:

  **I used 3µl of my template in 6µl rxns, and I cleaned them up using the ethanol

      precipitation protocol.