2TRNL Mule Deer

Extractions

• Transfer fecal material to labeled bead tubes. Weigh first few samples to approximate amount of material ~200mg
• Freeze dry samples
• Extract using MN Nucleospin 96 Stool kits following spin speeds from MN Nucleospin 96 Soil kits
• Transfer DNAs to plate
• Transfer 30 ul as working aliquot
• Add 4 samples of 5 ng/ul Mock Community Only and 4 samples of half mock community half DNA from a single plant sample DNA to column 9(first empty column) of 2TRNL8
• Add ISD and coligos
• Normalize

PCR MasterMix (28 Plates)

• Add 11 ul to each well of a hard shell, full skirt plate. Add 2 uL of primers and 2uL of template to each well.

Template Format:

Run TRNL plates on thermocycler program TRNL_T*:

MagBead Cleanup:

• Equilibrate Beads to room Temperature

• Add 15uL of ultra pure water to each well.

• Add 24 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 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 1:1000 dilutions of wells B3 & H6 from the PCR plate 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: