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
4. Cover the block only with the plastic lid that comes with it.
5. Grow overnight at 37°C shaking at 225rpm.
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.
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.
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
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.
**I used 3µl of my template in 6µl rxns, and I cleaned them up using the ethanol