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This experiment is to verify fullITS these CO1 primers can be sequenced. Primer Source Paper

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95 °C for 60 s, 45 °C for 90 s, 72 °C for 90 s; 28 cycles of: 94 °C for 60 s, 50 °C for 90 s, 72 °C for 60 s

PCR MasterMix

ul/rxn	Reagent	# of rxns	ul needed

3

5X Kapa HiFi Buffer

384

1320

0.45

10M dNTPs

384

198

0.3

7.5	Kapa HiFi HotStart DNA

Pol

2X

384

120

132

900

7

4.

25

5

HPLC H2O

384

120

3190

540

11

12

Total Volume

384

4840

1440

Add 11 ul to each well of a hard shell, full skirt plate. Add 2 uL of 0.5 uM primers and 2uL of template to each well.
Primers:

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Run CO1Lark plates on thermocycler program CO1Lark:

Step	Temp C	Cycles	Time
Denature	95	1X	10:00
Denature	94	35X	1:00
Annealing** (Row C)	50	35X	1:30
Extension/Elongation	72	35X	1:00
Final Extension	72	1X	10:00
Hold	4	1X	0:00

Pool duplicates together.

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Pool 2 ul of the TRNL and 16S samples separately. Make 1:1000 dilutions of each pool and run in triplicate.
Make 1:1000 dilutions of columns 1,6, and 12 for each plate using 999 of TE and 1uL of sample into a deep well plate.

	1	2	3	4	5	6	10	11	12
A	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12	NTC	NTC	NTC
B	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12	2 pM Std	2 pM Std	2 pM Std
G	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12	20 pM Std	20 pM Std	20 pM Std
H	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12

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)	110	1100
2 ul	Primer Premix (10X)	110	220
4 ul	Ultra Pure Water	110	440
16 ul	Total Volume	110	1760

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

	1	2	3	4	5	6	7	10	11	12
A	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12	TRNL1_1_16S_Pool	NTC	NTC	NTC
B	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12	TRNL1_1_16S_Pool	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12	TRNL1_1_16S_Pool	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12	TRNL1_1_T_Pool	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12	TRNL1_1_T_Pool	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12	TRNL1_1_T_Pool	2 pM Std	2 pM Std	2 pM Std
G	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12		20 pM Std	20 pM Std	20 pM Std
H	TRNL1_1_16S_Col1	TRNL1_1_16S_Col6	TRNL1_1_16S_Col12	TRNL1_1_T_Col1	TRNL1_1_T_Col6	TRNL1_1_T_Col12

Results:

The TRNL pool via standard size estimation returned a mean of ? nM. This should be adjusted for the difference between the standards' fragment sizes and the expected product size (452 vs 220). 9.41x(452/220) = 19.33 nM

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PCR MasterMix

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