LRII.5 Repeat equimolar pooling with the addition of more control samples

New Control Samples:

Start Reaction Plate Setup:

• Add Ultrapure H2O to the reaction plates in the following pattern:

NOTE: Water in the master mix has been adjusted to a lower amount. The plate set up below will replace the water usually added to the master mix while also showing if a decrease in water and an increase in template will increase PCR yields.

MasterMix

• Add 5 ul to each well of a hard shell, full skirt plate.

• Add 10 ng/ul Mock Community to the plate in the following pattern:

• Add 2 ul 1-step primers: long16S0A6

• Seal with bubble seals. Vortex briefly. Spin down.

• Run on Thermocycler Program GSAF36:

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)

qPCR all products:

• Make 1:1000 dilutions of all samples 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:

Redone equimolar pooling:

SAG_S30P2R_R_8uL, highlighted in red might not have 26.1 ul left in the well, just use the remainder. SAG_S38P1R_R_8uL, highlighted in yellow, does not have enough V left, so just omit from pool.

The green highlighted samples/wells should be diluted by adding 2 ul of the sample to 14 ul TE. The ul indicated in the last column should be added to the new pool fro this dilution.

Pool the new Mock Community samples to the above pool at 1 ul each.

iSeq Run:

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

1000/12.97 = 77uL of Pool to Add

• 1000 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add

1000- 77 = 923uL of 10mM Tris 8.5

Pool with newest 1 nM RM_Jan22 samples at a 1:2 ratio (1 part LowRead, 2 parts RM_Jan22)

Loading pool is:

80ul 10mM Tris HCL pH 8, 16 ul 50 pM PhiX, 4 ul from above pool.