Test Samples - BioAnalyzer (TRNL & AMF)

We will run 6 samples of TRNL (3 soil and 3 plant) as well as 6 samples of Bo Steven’s AMF samples at 50 cycles on the BioAnalyzer to make sure the PCR is optimized to obtain a sufficient amount of product.

PCR MasterMix

• Add 11 ul to 6 wells of two PCR strip tubes. Add 2 uL of primers and 2uL of template to each well.

• TRNL Primers: A1 Position: TRNL02, TRNL07, TRNL11, TRNL14, TRNL19, TRNL23 (Strip Tube 1)

• AMF Primers: AMF06 (Strip Tube 2)

Template Format:

Strip Tube 1: Run on thermocycler program TRNL_50:

Strip Tube 2: Run on thermocycler program AMF_50:

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)

BioAnalyzer:

• BioAnalyzer Chip Set up:

Results:

TRNL/AMF GEL:

Open

TRNL/AMF EPG:

Open

TRNL_PLANT_EPG:

TRNL_STOOL_EPG:

AMF_PLT1_EPG:

AMF_PLT2_EPG:

**Due to the discrepancy between the TRNL plant and stool samples, we will be conducting a second experiment to verify results.