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.

This run failed without detecting any clusters. So, we are repeating it.

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.