Page 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.

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

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

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

Cleanup of PCR 1 product:

• This was done on the Nimbus platform using “AxyPrep MagBead PCR1 No MM”

  and manually. Probably 3/4 of the plates were cleaned manually.

• Manual protocol

• Equilibrate Beads to room Temperature

  • 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 30 ul H2O; 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 30uL to clean transparent PCR plate.

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Final Cleanup:

Equilibrate Beads to room Temperature

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