2AMF Library Prep

Plates: 2AMF1 and 2AMF2

	1	2	3	4	5	6	7	8	9	10	11	12	1

Remaining 2 columns are Mock Community

PCR MasterMix

ul/rxn	Reagent	# of rxns	ul needed

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	530	1590
0.45	10M dNTPs	530	238
0.3	Kapa HiFi HotStart DNA Pol	530	159
7.25	HPLC H2O	530	3843
11	Total Volume	530	5830

Add 11 ul to each well of a hard shell, full skirt plate. Add 2 uL of primers and 2uL of template to each well.

Plate	18S Primer	ITS

Run AMF on thermocycler program AMF35:

Step	Temp C	Cycles	Time

Step	Temp C	Cycles	Time
Denature	95	1X	10:00
Denature	95	35X	0:30
Annealing	55	35X	0:30
Extension/Elongation	72	35X	1:00
Extension/Elongation	72	1X	9:00
Hold	4	1X	0:00

And ITS on GSAF36

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)

Make 1:1000 dilutions of column 4 or 2 and 4 (2AMF2) from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:

	1	2	3	4	5	6	7	8	9	10	11	12