4AH1 & GC2017-2 1Step vs 2Step PCR Comparison

We will be comparing the difference in results for 4AH1 using the 2 step PCR and the 1 step PCR. The 1 step PCR protocol used for 4AH1 is under the page named “NovaSeq4 1Step PCR and qPCR”. Below is the protocol used for the 2 step PCR. We will also be repeating the 2 step for PCR for GC2017-2.

*DO NOT USE THE FOLLOWING 2 STEP PCR MID PLATES (16S OR ITS) FOR 4AH1: A2, B2, A4, B4, C8, D8

4AH1 PCR1 MasterMix (make for 4 plates in 5mL tube)

• Add 7 ul to each well of 16 hard shell, full skirt plate. Add 2 ul of template and 6 ul of the primers.

• Spin down resulting “PCR1”s and then add to thermocyclers running “35GSAF1”

• Store at 4C when cycler reaches below 40C unless immediately proceeding to next step.

Cleanup of PCR 1 product

• Equilibrate Beads to room Temperature

  • Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate

  • 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)

4AH2 PCR2 MasterMix (make for 2 plates in 1.5mL tube)

Add 5 ul master mix to all wells of 2 hard shell full skirted plates. Seal with tape seals and store in refrigerator if not using immediately. Add 10ul of template from PCR1. Run PCR2 on 35GSAF2 program.

Final Cleanup:

• Equilibrate Beads to room Temperature

  • Add 15 ul H2O to each sample

  • 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 54 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 per GSAF protocol; pipette mix 10 times

  • Incubate at RT for 2 minutes

  • Place sample plate back on magnet for 5 minutes or until all wells are cleared.

  • Transfer 40 ul to a clean transparent PCR plate.

Run on qPCR:

Column 3 of 4AH1_16S and 4AH1_ITS was used for quantification.

4AH1_16S: 3.71 nanomoles

4AH1_ITS: 13.58 nanomoles

The full result report can be viewed below:

Open

GC2017-2 PCR1 MasterMix (make for 4 plates in 5mL tube)

• Add 7 ul to each well of 16 hard shell, full skirt plate. Add 2 ul of template and 6 ul of the primers.

• Spin down resulting “PCR1”s and then add to thermocyclers running “35GSAF1”

• Store at 4C when cycler reaches below 40C unless immediately proceeding to next step.

Cleanup of PCR 1 product

• Equilibrate Beads to room Temperature

  • Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate

  • 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)

GC2017-2 PCR2 MasterMix (make for 2 plates in 1.5mL tube)

Add 5 ul master mix to all wells of 2 hard shell full skirted plates. Seal with tape seals and store in refrigerator if not using immediately. Add 10ul of template from PCR1. Run PCR2 on 35GSAF2 program.

Final Cleanup:

• Equilibrate Beads to room Temperature

  • Add 15 ul H2O to each sample

  • 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 54 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 per GSAF protocol; pipette mix 10 times

  • Incubate at RT for 2 minutes

  • Place sample plate back on magnet for 5 minutes or until all wells are cleared.

  • Transfer 40 ul to a clean transparent PCR plate.

Run on qPCR:

Column 3 of GC2017-2_16S and GC2017-2_ITS was used for quantification.

GC2017-2_16S: 25.61

GC2017-2_ITS: 26.54

The full result report can be viewed below:

Open