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Info
Data was returned on 9-21-2021. Data was transferred to client and invoiced. M01247

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”

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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:

View file

name	5CM_HMAXPP_5RM1.csv

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)

Fragment Analysis returned from the Genomics Core lead us to allow sequencing to proceed.

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Well	Conc pg/ul	Sample Description
EL1	2350	Electronic Ladder
A1	2380	18-143T
B1	1090	SB33N
C1	1390	18-143N
D1	1450	51N
E1	688	excontrol
F1	1890	5CM pool

Versions Compared

Old Version 4

New Version Current

Key

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

MagBead Cleanup:

Results:

Pooling and Shipping: