CEAg 16S 2026 (26MIJA1)

MasterMix

*Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label. (If doing manually, one needs 2 ul of template and 2 ul of the primers).

Used long16S0H12

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

• Make a 1:1000 dilution of HMax_PP Final Pool to check quality of pippin elution before sending out for RFS.

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

26WARD1 4 nM

26MIJA1 3 nM

Dilute 26WARD to 1 nM: 25 ul sample with 75 ul RSB

Dilute 26MIJA to 1 nM: 34 ul sample with 66 ul RSB

Combine to 400 pM: 2.5 ul MIJA with 47.5 ul ul WARD and 75 ul RSB

Pooling and Shipping: