Jorgenson Test PCR

Template: 2 ul of 10ng/ul Zymo Mock Community or Karen Jorgenson’s Test DNAs

• Add 11 ul to each well of 2 hard shell, full skirt plates, Seal with bubble strips, store in refrigerator until needed labeled “PCR1”.

• Add 2 ul template

• Add 2 ul primers (16S0H8 and ITS0H8)

Run on Thermocycler Program GSAF36:

Purify samples using GSAF’s modified MagBead protocol:

Equilibrate Beads to room Temperature

Combine ITS sample with 16S

Add 24 ul (0.8 x 60 ul) of MagBeads to each well; 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 108 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.

Allow plate to air dry for 7 minutes.

Remove sample plate from magnet plate.

Add 40 ul TE to reach sample

Incubate at RT for 2 minutes

Place sample plate back on magnet for 2 minutes or until all wells are cleared.

Transfer 40 ul to a clean PCR plate or wells.

qPCR:

Pool of 2 ul from each well then dilute 1 ul in 999 ul H2O.

Dilute 1 ul of the first 3 columns in 999 ul H2O.

Run on qPCR

qPCR Results:

All of Karen’s PCRed samples amplified to above minimums for all Illumina platforms with which I (Gregg R) have dealt. The largest requirement is for the NovaSeq at 1 nM. Hers were all above 1.5 nM.

Results