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)
...
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00 |
98 | 20X | 0:30 |
55* | 20X | 0:30 |
72 | 20X | 0:30 |
72 | 1X | 5:00 |
4 | 1X | 0:00 |
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.