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)
...
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.
...
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.
...