Amplicon MasterMix Prep and QC protocol

MasterMix (make for 40 plates in 50mL conical tube)

Take out 6 tubes of dNTPs and 9 tubes of 5x Buffer to thaw.

Get new 50 ml tube from pre-PCR side. Volumetric dispense 30 ml from Water Filer system. Remove 275 ul to reach 29725 ul. Label tube.

Take out 5 tubes of Taq when ready to assemble.

Aliquot 4,490 ul into 10 5ml tubes and label each A_mmddyy_AmpliconStdMM_#

Aliquot 11 ul into each well of an 8 pcr-tube strip. Add 4 ul IlluminaTest primers to 2. Add 4 ul 16STest primers to 1; add 2 ul 16STest primers to the next and 2 ul TE. Add 4 ul ITSTest primers to 1; add 2 ul ITSTest primers the next and 2 ul TE., Add 2 ul 16STest primers and 2 ul MC to the last 2. Add Test date to TE source.

Cap, Vortex, and spin down. Run on thermocycler program GSAF36:

No cleanup needed. Run resultant rxns on a D1000 High Sensitivity Tape.

• Remove reagents and tape out of fridge for 30 minutes

• Vortex and spin down Buffer and Ladder

• Add 2 ul High Sensitivity Buffer to 9 wells of 2 empty Agilent Optical Tube Strips

• Add 2 ul Ladder to the first well of one.

• Add 2 ul sample to each well you added Buffer.

• Apply caps, Label tops, and quick spin down.

• Transport to INBRE.

• Open TapeStation Software

• Add Tape to slot with QR code in the back right

• Vortex tube strips for 1 minute at 2000 rpm

  • Assign sample names in software

• Spin tubes strips down for 1 minute

• Add samples info instrument with ladder in A1 (back left)

• After run completion, Create Report and add to report subpage

  • email or use usb to transfer data

• After use, save any remaining MM in 2 ml tube. Transfer appropriate label for post sequencing check if necessary.