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Version	Old Version 2	New Version Current
Changes made by	Gregg Randolph	Gregg Randolph
Saved on	Aug 03, 2022	Jan 21, 2023

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Plates: 2AMF1 and 2AMF2

	1	2	3	4	5	6	7	8	9	10	11	12	1
A	2	23	34	44	54	63	71	92	185	193	201	209	216
B	6	24	36	45	55	64	72	178	186	194	202	210	217
C	15	26	38	46	56	65	85	179	187	195	203	211	218
D	16	28	39	47	57	66	86	180	188	196	204	212	219
E	19	29	40	48	58	67	87	181	189	197	205	213	220
F	20	30	41	50	59	68	88	182	190	198	206	214	221
G	21	32	42	51	61	69	90	183	191	199	207	215	222
H	22	33	43	52	62	70	91	184	192	200	208	Blank	Blank2

Remaining 2 columns are Mock Community

PCR MasterMix

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

...

Plate	18S Primer	ITS
2AMF1	AMF01	ITS0A9
2AMF1	AMF02	ITS0B9
2AMF2	AMF03AMF04AMF04	ITS0C9
2AMF2

Run AMF on thermocycler program AMF35:

...

Make 1:1000 dilutions of column 1,6,10 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
A	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS						NTC	NTC	NTC
B	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS						0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS						0.002 pM Std	0.002 pM Std	0.002 pM Std
D	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS						0.02 pM Std	0.02 pM Std	0.02 pM Std
E	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS						0.2 pM Std	0.2 pM Std	0.2 pM Std
F	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS						2 pM Std	2 pM Std	2 pM Std
G	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS						20 pM Std	20 pM Std	20 pM Std
H	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS

Add 16 ul of Illumina Library Quantification MasterMix to each well:

...

Add 4 ul of template, pool, or standards to each well:

	1	2	3	4	5	6	7	8	9	10	11	12
A	BS2AMF1_PLT1AMF	2AMF1_Col1ITS	BS2AMF2_PLT1_Col6AMFBS	2TRNL_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10	BS_AMF_Pool		NTC16S	40 16S	40 16S	40 16S	40 ITS	40 ITS	40 ITS	NTC	NTC
B	BS2AMF1_PLT1_Col1AMFBS	2AMF1_PLT1_Col6ITSBS	2AMF2_PLT1_Col10AMFBS	2TRNL_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10	BS_AMF_Pool			0.0002 pM Std16S	4 16S	4 16S	4 16S	4 ITS	4 ITS	4 ITS	0.0002 pM Std	0.0002 pM Std
C	BS2AMF1_PLT1_Col1AMFBS	2AMF1_PLT1_Col6ITSBS	2AMF2_PLT1_Col10AMFBS	2TRNL_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10	BS_AMF_Pool			0.002 pM Std16S	0.4 16S	0.4 16S	0.4 16S	0.4 ITS	0.4 ITS	0.4 ITS	0.002 pM Std	0.002 pM Std
D	BS2AMF1_PLT1_Col1AMFBS	2AMF1_PLT1_Col6ITSBS	2AMF2_PLT1_Col10AMFBS	2TRNL_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10				0.02 pM StdT	0.04 16S	0.04 16S	0.04 16S	0.04 ITS	0.04 ITS	0.04 ITS	0.02 pM Std	0.02 pM Std
E	BS2AMF1_PLT1_Col1AMFBS	2AMF1_PLT1_Col6ITSBS	2AMF2_PLT1_Col10ITSBS	2TRNL_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10				0.2 pM StdT	0.004 16S	0.004 16S	0.004 16S	0.004 ITS	0.004 ITS	0.004 ITS	0.2 pM Std	0.2 pM Std
F	BS2AMF1_PLT1_Col1AMFBS	2AMF1_PLT1_Col6ITSBS	2AMF2_PLT1_Col10ITSBS	2TRNL_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10				2 pM StdT	0.0004 16S	0.0004 16S	0.0004 16S	0.0004 ITS	0.0004 ITS	0.0004 ITS	2 pM Std	2 pM Std
G	BS2AMF1_PLT1_Col1AMFBS	2AMF1_PLT1_Col6ITS	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10	2AMF2_ITS								20 pM Std	20 pM Std	20 pM Std
H	BS2AMF1_PLT1_Col1AMFBS	2AMF1_PLT1_Col6ITS	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10	2AMF2_ITS

Results:

View file

name	BS_AMF.csv

...