2-step re-prep of sm19-fire (5RD1)

PCR1 (Manual):

• Create MasterMix:

• Add 7 ul to each well of 6 hard shell, full skirt plates, Seal with tape seals, rub with kimwipe across all wells twice, and then around the edge, store in refrigerator until needed labeled “PCR1”

• Spin down 4 “PCR1” plates. Vortex and spin 1 “_Norm” plate. Vortex and spin to 16S MID plates and 2 ITS MID plates.

• Remove the seals from the 4 “PCR1” plates. Attach tips from a freshly opened 20 ul tip box TO THE BENCHSMART. Add 2 ul to a “PCR1” plate.

• Dispose of these tips.

• Grab 1 new 20 ul tip box. Cover 3 of the “PCR1” plates. Grab one of the MID plates.

• Remove the bubble caps from the appropriate MID plate.

• Transfer 6 ul to the corresponding column of the “PCR1” plate. Recap the MID plate and cap the resulting plate.

• Repeat the previous 5 steps matching each “PCR1” plate with a unique MID plate.

• Move “_Norm” plate to -80 storage. Move MID plates to “Used MIDs” storage section.

• Spin down resulting “PCR1”s and then add to thermocyclers running “35GSAF1”

PCR2 Mastermix Preparation:

Add 5 ul master mix to all wells of 4 hard shell full skirted plates. Seal with tape seals and store in refrigerator.

Intermediate Cleanup:

The majority of this was done on the Nimbus platform using “AxyPrep MagBead PCR1 No MM”

Manually, it was done:

• Equilibrate Beads to room Temperature

• Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate

• Pipette mix up and down 10 times.

• Incubate at RT for 5 minutes

• Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)

• Remove 65 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.

• Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well

• Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well

• Reaspirate from each well to assure maximum EtOH removal

• Allow plate to air dry for 7 minutes.

• Remove sample plate from magnet plate.

• Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.

• Place sample plate back on magnet for 5 minutes or until all wells are cleared.

• Transfer 40 ul to labeled transparent plate (Plate1 PCR1 MIDPlate1 MIDPlate2)

• Transfer 10 ul from transparent plate to “PCR2” plate with Mastermix already added.

• Label Plate1 PCR2 MIDPlate1 MIDPlate2

• Seal “PCR2”s with bubble strips and run on thermocycler 35GSAF2 program

*PCR2 actually had 20 cycles, not 19

Final Cleanup:

Equilibrate Beads to room Temperature

Add 15 ul H2O to each sample

Add 24 ul (0.8 x 60 ul) of MagBeads to each well; Pipette mix up and down 10 times

Incubate at RT for 5 minutes

Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)

Remove 54 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.

Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.

Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.

Allow plate to air dry for 7 minutes.

Remove sample plate from magnet plate.

Add 40 ul TE per GSAF protocol; pipette mix 10 times

Incubate at RT for 2 minutes

Place sample plate back on magnet for 5 minutes or until all wells are cleared.

Transfer 40 ul to a clean transparent PCR plate labeled “Plate1 PCR2 MIDPlate1 MIDPlate2