BigHornSheep 16S 2021 (5CM)

5CM1 (16S only)	5CM2 (16S only)	5CM3 (16S only)	5CM4 (16S only)

MasterMix (make for 8 plates in 15mL conical tube)

ul/rxn	Reagent	# of rxns	ul needed (1.5X)
3	5X Kapa HiFi Buffer	768	3456
0.45	10M dNTPs	768	518.4
0.3	Kapa HiFi HotStart DNA Pol	768	345.6
7.25	HPLC H2O	768	8352
11	Total Volume	768	12672

*Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “NovaSeq4 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).

Plate	16S MID	ITS MID
5CM1	16S0A1	N/A
5CM1	16S0B1	N/A
5CM2	16S0C1	N/A
5CM2	16S0D1	N/A
5CM3	16S0E1	N/A
5CM3	16S0F1	N/A
5CM4	16S0G1	N/A
5CM4	16S0H1	N/A

Run on Thermocycler Program GSAF36:

Temp C	Cycles	Time
95*	1X	3:00*
98	36X	0:30
62	36X	0:30
72	36X	0:30
72	1X	5:00
4	1X	0:00

MagBead Cleanup:

Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”

Manually, it was done:

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)

qPCR NovaSeq5_Plate1

Make 1:1000 dilutions of column 3 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:

	1	2	3	4	10	11	12
A	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3	NTC	NTC	NTC
B	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3	2 pM Std	2 pM Std	2 pM Std
G	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3	20 pM Std	20 pM Std	20 pM Std
H	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3

Make a 1:1000 dilution of HMax_PP Final Pool to check quality of pippin elution before sending out for RFS.
Add 16 ul of Illumina Library Quantification MasterMix to each well:

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	60	600
2 ul	Primer Premix (10X)	60	120
4 ul	Ultra Pure Water	60	240
16 ul	Total Volume	60	960

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

	1	2	3	4	5	10	11	12
A	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3	HMax_PP_Final	NTC	NTC	NTC
B	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3	5RM1_1nm_Pool	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3		0.002 pM Std	0.002 pM Std	0.002 pM Std
D	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3		0.02 pM Std	0.02 pM Std	0.02 pM Std
E	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3		0.2 pM Std	0.2 pM Std	0.2 pM Std
F	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3		2 pM Std	2 pM Std	2 pM Std
G	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3		20 pM Std	20 pM Std	20 pM Std
H	5CM1_16S_Col3	5CM2_16S_Col3	5CM3_16S_Col3	5CM4_16S_Col3

Results:

Average results for the following plates (column 3):

5CM1_16S: 6.03 nanomoles

5CM2_16S: 9.32 nanomoles

5CM3_16S: 3.52 nanomoles

5CM4_16S: 9.77 nanomoles

Results for HMax_PP_Final can be viewed in result report below and have been added to the HMax page here.

Full result report can be viewed below:

Pooling and Shipping:

The client, Chris MacGlover, is concerned about the nasal swabs not sequencing as occurred with his last attempt. We will send 3 nasal, one excontrol, and one other sample to be fragment analyzed at CU:

5CM1 A4 (18-143N), 5CM1 H12 (SB33N), 5CM2 A4 ( 18-143T), 5CM3 H12 (51N), and 5CM1 A3 (excontrol)