ReProcess 5FB1 1-Step PCR and qPCR

Master Mixes used with links to QC pages

10-20-22_AmpliconMM : QC

PCR

• Pull Out 5FB1_Norm and allow to thaw; wash hands before proceeding.

• Pull Out 4 hard shell, full skirt plates and label them 7-5FB1_16S1, 7-5FB1_16S2, 7-5FB1_ITS1, and 7-5FB1_ITS2

• Pull Out 10-20-22_AmpliconMM_1 and allow to thaw

• Pull Out long16S0C1, long16S0D1, longITS0C1, and longITS0D1 and vortex and spin down

• Add 11 ul MM to each well of the 4 hard shell, full skirt plates. Save MM remainder in a clean 2 ml tube. Transfer label.

• Add 2 ul 5FB1 Norm to each plate

• Add 2 ul from one of the primer plates to each plate. Seal, vortex, and spin.

Run on Thermocycler Program GSAF36:

• Remember to save any remaining MM in a 2 ml tube, transfer label from 5 ml, and return to -20 for possible future retesting.

MagBead Cleanup:

Some plates were cleaned up using the Nimbus platform protocol “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 (Plate name_PCR_MIDs)

• Store in -20 until ready to qPCR

qPCR NovaSeq5_ConTest1

• Make 1:1000 dilutions of column 8 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:

• Add 16 ul of Illumina Library Quantification MasterMix to each well:

• Add 4 ul of template, pool, or standards to each well:

Results:

Full qPCR results below:

 Sequencing Test:

Dilute to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.

• 1000/Results = ul of Pool to Add

100/8.4 = 12uL of Pool to Add

• 100 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add

100- 12 = 82 uL of 10mM Tris 8.5

Dilute 1 nM full pool to loading concentration of 60 pM:

• Add 6 ul 1 nM Pool to 82 ul “10 mM Tris 8.5” and 12 ul 50 pM PhiX

• Remove iSeq 100 i1 Flow Cell from refrigerator 5’s crisper drawer and open white foil pack and allow to equilibrate to RT for 10-15 minutes.

• Open “iSeq 100 i1 Reagent Cartridge v2”. Turn on iSeq100

• Click on “Sequence”. Watch Video. Do what video tells you to do. Follow on screen instructions until run starts.

Redone Sequencing Test:

Dilute to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.

• 1000/Results = ul of Pool to Add

100/10 = 10uL of Pool to Add

• 100 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add

100- 10 = 90 uL of 10mM Tris 8.5

Dilute 1 nM full pool to loading concentration of 60 pM:

• Add 6 ul 1 nM Pool to 82 ul “10 mM Tris 8.5” and 12 ul 50 pM PhiX

• Remove iSeq 100 i1 Flow Cell from refrigerator 5’s crisper drawer and open white foil pack and allow to equilibrate to RT for 10-15 minutes.

• Open “iSeq 100 i1 Reagent Cartridge v2”. Turn on iSeq100

• Click on “Sequence”. Watch Video. Do what video tells you to do. Follow on screen instructions until run starts.