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Do an annealing temperature gradient for PCR (Row D will be suggested and then do up and down temperature from there).

Row A - 6 plants, 6 stools copied down to each row (full plate) see setup belowTest samples were previously extracted.

1. Make sample spreadsheet and plate test samples by adding 30uL of sample to the corresponding well.

2. Quantify samples on Synergy HTX.

3. Normalize samples to 10ng/uL.

4. Choose 4 plant and 4 stool to run PCR test.

5. Make 1 step PCR MMX and plate in the template format below.

PCR MasterMix

• 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.

• Primers: TRNL01

Template Format:

Run on thermocycler program TRNL_T*:

*TRNL_T program will test the efficiency of the PCR/annealing temperature by running it on a temperature gradient for each row. The table below shows the annealing temperature for each row.

**

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 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 TRNL_Test

• Make 1:1000 dilutions of column 1-8 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:

Results Straight Off ABI 7500

Average results