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Plates: BS_PLT1, BS_PLT2

PCR MasterMix

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	390	1170
0.45	10M dNTPs	390	176
0.3	Kapa HiFi HotStart DNA Pol	390	117
7.25	HPLC H2O	390	2827
11	Total Volume	390	4290

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

Plate	Primer
BS_PLT1	AMF01
BS_PLT1	AMF02
BS_PLT2	AMF03
BS_PLT2	AMF04

Run on thermocycler program AMF35:

Step	Temp C	Cycles	Time
Denature	95	1X	10:00
Denature	95	35X	0:30
Annealing	55	35X	0:30
Extension/Elongation	72	35X	1:00
Extension/Elongation	72	1X	9:00
Hold	4	1X	0:00

MagBead Cleanup:

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

Make 1:1000 dilutions of column 1,4,8,11 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:

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

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	8	9	10	11	12

	1	2	3	4	5	6	7	8	10	11	12
A	S1_A	S2_A	S3_A	S4_A	S5_A	S6_A	S7_A	S8_A	NTC	NTC	NTC
B									0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C									0.002 pM Std	0.002 pM Std	0.002 pM Std
D									0.02 pM Std	0.02 pM Std	0.02 pM Std
E									0.2 pM Std	0.2 pM Std	0.2 pM Std
F									2 pM Std	2 pM Std	2 pM Std
G									20 pM Std	20 pM Std	20 pM Std
H

Results:

There were some outliers, but most samples generated 10s of nano moles (~50).

The pool via standard size estimation returned a mean of 42.66 nM. This should be adjusted for the difference between the standards' fragment sizes and the expected product size (452 vs 481). 42.66x(452/481) = 40.087 or ~40 nM

Sequencing Test:

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

1000/40 = 25uL of Pool to Add

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

1000- 25 = 975 uL of 10mM Tris 8.5

Dilute 1 nM full pool to loading concentration of 50 pM:

Add 5 ul 1 nM Pool to 85 ul “10 mM Tris 8.5” and 10 ul 50 pM PhiX
Remove iSeq 100 i1 Flow Cell from refrigerator 5’s crisper drawer and open white foil pack and allow to equilibrate to RT for 10-15 minutes.
Open “iSeq 100 i1 Reagent Cartridge v2”. Turn on iSeq100
Click on “Sequence”. Watch Video. Do what video tells you to do. Follow on screen instructions until run starts.