Final QC from Psomagen

Final Closeup of Electropherogram from Psomagen

Sequencing Report from Psomagen:

Demultiplex Key. R-Script for Key.

Sample Input Sheet

Spreadsheet of samples and their current status.

Initial Sample Processing for Tubes:

1. Tubes were transferred from their initial container/box/rack to a lidded 96 well 2ml tube rack arrayed like a plate with corresponding row and column assignments labeled.

2. Racks were assigned plate names on a piece of lab tape

3. Tubes were vortexed and spun quickly and transferred to a new rack by column, along with the plate/rack label.

4. 30 ul was transferred, one column of 8 at a time, from the tubes to a labeled transparent PCR plate using a 50 ul expandable spacing pipette

5. Tubes were column wise systematically transferred to an identical rack and scanned or transcribed into a Google Sheet.

Initial Sample Processing for Plates:

Some plates were provided as aliquots. Others were not. Those that were not had 30 ul transferred to a labeled transparent PCR plate. Most plates got assigned a unique alternate plate name for ease of tracking and transfer through the processing, others came with appropriately short names assigned.

Shared Secondary Sample Processing:

1. 6 ul of the control plate (after vortex and spin) was added to each sample aliquot column wise with each well getting the well matching coligo. An asterisk was added to the plate to denote this.

2. Each plate was then quantified via absorbance using the HTX plate reader and its Take 3 Trio; elution buffer was used to blank the system. A circle was added around the asterisk to denote this.

3. Each resulting file was used to create a Normalization Worksheet.

4. This was then used to inform the Nimbus program “Normalization_Tracking” to create a separate normalized plate. The short plate name was added to the new transparent plate with the appendix “_Norm”

   1. plates were normalized to 10 ng/ul

   2. some plates that had all samples below this concentration were not normalized prior to the next step

5. The resulting “_Norm” plates were used to setup PCR either on the Nimbus platform or by hand.

PCR1 (Manual):

• Create MasterMix:

• Add 7 ul to each well of 8 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 the first column of tips from a freshly opened 10 ul tip box. Add 2 ul to the top, left side of each “PCR1” plate’s column one.

• Dispose of these tips. Attach the second column of tips. Add 2 ul to the top, left side of each “PCR1” plate’s column two. Repeat step until all tips are consumed. Reseal “_Norm”.

• Grab 4 new 10 ul tip boxes. Cover 3 of the “PCR1” plates. Grab one of the MID plates.

• Remove the bubble caps from the first column of the MID plate with your left hand by the numbered side.

• Attach the first column of tips to your 8 channel. Transfer 6 ul to the corresponding column of the “PCR1” plate. Return the caps in the same orientation they were removed.

• Repeat previous steps until all tips are used. Add bubble caps to the “PCR1” plate. Add the base plate name from the “_Norm” plate and the barcode of the MID plate to the “PCR1” plate.

• Repeat the previous 3 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”

PCR1(automated):

• Ensure Nimbus and laminar flow are turned on.

• Collect 1 “_Norm” plate, 4 “PCR1” MM plates, and 2 16S MID plates and 2 ITS MID plates. Label all PCR 1 plates with the base name of the “_Norm” plate and one of the MID plates.

• Open ““ in Nimbus “Run Protocol” window. Press green triangle. Remove seals and layout the plates and tips out according to the layout that pops up. Follow on screen prompts.

• Remove plates after“Method Completed” pops up. Reseal. Seal “PCR1”s with bubble strips. Spin down and run on thermocycler as above. Leave in thermocycler until Lid Temp drops below 40C to avoid cap warping and evaporation.

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; 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 30 ul H2O; 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 30 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