Page Comparison

New Control Samples:

...

	1	2	3	4	5	6	7	8	9	10	11	12
A	RA6	RA14	RA22	LL1A_J	WG3A_S	LY2A_S	MCA_2	MCA_4	MCA_8	NTC	NTC	NTC
B	RA7	RA15	RA23	LL1B_J	WG3B_S	LY2B_S	MCB_2	MCB_4	MCB_8	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	RA8	RA16	RA24	LL2A_J	WG4A_S	LY3B_S	MCC_2	MCC_4	MCC_8	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	RA9	RA17	RA25	LL2B_J	WG4B_S	LY1A_S	MCD_2	MCD_4	MCD_8	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	RA10	RA18	RA26	LL4A_J	WG2A_S	LY1B_S	MCA_2_clean	MCA_4_clean	MCA_8_clean	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	RA11	RA19	RA27	LL4B_J	WG2B_S	LY4A_S	MCB_2_clean	MCB_4_clean	MCB_8_clean	2 pM Std	2 pM Std	2 pM Std
G	RA12	RA20	RA28	LL3A_J	WG1A_S	LY4B_S	MCC_2_clean	MCC_4_clean	MCC_8_clean
H	RA13	RA21	RA29	LL3B_J	WG1B_S	BLANK	MCD_2_clean	MCD_4_clean	MCD_8_clean
Plate	5LVD5	5LVD5	5LVD5	5DG5	5DG5	5DG5	Controls	Controls	Controls
Column	1	2	3	4	5	6

...

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	80110	8001100
2 ul	Primer Premix (10X)	80110	160220
4 ul	Ultra Pure Water	80110	320440
16 ul	Total Volume	80110	12801760

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

	1	2	3	4	5	6	7	8	9	10	11	12
A	RA6	RA14	RA22	RA6	RA14	RA22	RA6	RA14	RA22	NTC	NTC	NTC
B	RA7	RA15	RA23	RA7	RA15	RA23	RA7	RA15	RA23	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	RA8	RA16	RA24	RA8	RA16	RA24	RA8	RA16	RA24	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	RA9	RA17	RA25	RA9	RA17	RA25	RA9	RA17	RA25	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	RA10	RA18	RA26	RA10	RA18	RA26	RA10	RA18	RA26	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	RA11	RA19	RA27	RA11	RA19	RA27	RA11	RA19	RA27	2 pM Std	2 pM Std	2 pM Std
G	RA12	RA20	RA28	RA12	RA20	RA28	RA12	RA20	RA28	20 pM Std	20 pM Std	20 pM Std
H	RA13	RA21	RA29	RA13	RA21	RA29	RA13	RA21	RA29

	1	2	3	4	5	6	7	8	9	10	11	12
A	LL1A_J	WG3A_S	LY2A_S	LL1A_J	WG3A_S	LY2A_S	LL1A_J	WG3A_S	LY2A_S	NTC	NTC	NTC
B	LL1B_J	WG3B_S	LY2B_S	LL1B_J	WG3B_S	LY2B_S	LL1B_J	WG3B_S	LY2B_S	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	LL2A_J	WG4A_S	LY3B_S	LL2A_J	WG4A_S	LY3B_S	LL2A_J	WG4A_S	LY3B_S	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	LL2B_J	WG4B_S	LY1A_S	LL2B_J	WG4B_S	LY1A_S	LL2B_J	WG4B_S	LY1A_S	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	LL4A_J	WG2A_S	LY1B_S	LL4A_J	WG2A_S	LY1B_S	LL4A_J	WG2A_S	LY1B_S	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	LL4B_J	WG2B_S	LY4A_S	LL4B_J	WG2B_S	LY4A_S	LL4B_J	WG2B_S	LY4A_S	2 pM Std	2 pM Std	2 pM Std
G	LL3A_J	WG1A_S	LY4B_S	LL3A_J	WG1A_S	LY4B_S	LL3A_J	WG1A_S	LY4B_S	20 pM Std	20 pM Std	20 pM Std
H	LL3B_J	WG1B_S	BLANK	LL3B_J	WG1B_S	BLANK	LL3B_J	WG1B_S	BLANK

	1	2	3	4	5	6	7	8	9	10	11	12
A	MCA_2	MCA_4	MCA_8	MCA_2	MCA_4	MCA_8	MCA_2	MCA_4	MCA_8	NTC	NTC	NTC
B	MCB_2	MCB_4	MCB_8	MCB_2	MCB_4	MCB_8	MCB_2	MCB_4	MCB_8	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	MCC_2	MCC_4	MCC_8	MCC_2	MCC_4	MCC_8	MCC_2	MCC_4	MCC_8	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	MCD_2	MCD_4	MCD_8	MCD_2	MCD_4	MCD_8	MCD_2	MCD_4	MCD_8	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	MCA_2_clean	MCA_4_clean	MCA_8_clean	MCA_2_clean	MCA_4_clean	MCA_8_clean	MCA_2_clean	MCA_4_clean	MCA_8_clean	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	MCB_2_clean	MCB_4_clean	MCB_8_clean	MCB_2_clean	MCB_4_clean	MCB_8_clean	MCB_2_clean	MCB_4_clean	MCB_8_clean	2 pM Std	2 pM Std	2 pM Std
G	MCC_2_clean	MCC_4_clean	MCC_8_clean	MCC_2_clean	MCC_4_clean	MCC_8_clean	MCC_2_clean	MCC_4_clean	MCC_8_clean	20 pM Std	20 pM Std	20 pM Std
H	MCD_2_clean	MCD_4_clean	MCD_8_clean	MCD_2_clean	MCD_4_clean	MCD_8_clean	MCD_2_clean	MCD_4_clean	MCD_8_clean
	Controls	Controls	Controls	Controls	Controls	Controls	Controls	Controls	Controls

...

Pool 2 ul from all original product plates. Make 3 1:1000 dilutions of pool and qPCR as usual. Dilute pool to 1nM. Dilute to loading concentration with 20% PhiX.

qPCR Master Mix:

Add 16 ul of Illumina Library Quantification MasterMix to each well of 4 plates:

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	40	800
2 ul	Primer Premix (10X)	40	160
4 ul	Ultra Pure Water	40	320
16 ul	Total Volume	40	1280

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

	1	2	10	11	12
A	LRIII_MC_MB_Pool1	LRIII_MC_MB_Pool3	NTC	NTC	NTC
B	LRIII_MC_MB_Pool1		0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	LRIII_MC_MB_Pool1		0.002 pM Std	0.002 pM Std	0.002 pM Std
D	LRIII_MC_MB_Pool2		0.02 pM Std	0.02 pM Std	0.02 pM Std
E	LRIII_MC_MB_Pool2		0.2 pM Std	0.2 pM Std	0.2 pM Std
F	LRIII_MC_MB_Pool2		2 pM Std	2 pM Std	2 pM Std
G	LRIII_MC_MB_Pool3		20 pM Std	20 pM Std	20 pM Std
H	LRIII_MC_MB_Pool3

Results:

View file

name	LRIII_MC_MB.csv

iSeq Run:

Dilute to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.

1000/Results = ul of Pool to Add

1000/73 = 13.7uL of Pool to Add

1000 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add

1000- 13.7 = 986.3 uL of 10mM Tris 8.5

Dilute to loading concentration with 20% PhiX.

Loading pool is:

75ul 10mM Tris HCL pH 8, 20 ul 50 pM PhiX, 5 ul from above pool.

Version	Old Version 4	New Version 5
Changes made by	Shannon Harris	Shannon Harris
Saved on	Mar 16, 2022	Mar 17, 2022

Versions Compared

Key

New Control Samples:

qPCR Master Mix:

Results:

iSeq Run: