Sequencing Test for Coligo Addition

We are going to prep and sequence on the iSeq Alfalfa TN plates 5 and 12 to make sure coligos were added to all plates that were foil sealed. Directions said to foil seal after coligo addition but due to the prolonged pause on this project, and no notes/plate notations revealing coligo addition we cannot be certain. Plates were checked for volume which did indicate coligo addition, but we will be sure through sequencing so we do not waste time/resources on PCR if coligos are not present.

MasterMix (make for 8 plates)

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	850	2,550
0.45	10M dNTPs	850	382.5
0.3	Kapa HiFi HotStart DNA Pol	850	255
7.25	Ultra Pure H2O	850	6,162.5
11	Total Volume	850	9,350

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

Plates	16S MIDs	ITS MIDs
ALF_PLT5_TN	long16S0C8	longITS0C8
ALF_PLT5_TN	long16S0D8	longITS0D8
ALF_PLT12_TN	long16S0E9	longITS0E9
ALF_PLT12_TN	long16S0F9	longITS0F9

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 ALF_END

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

	1	2	3	4	5	6	7	8	11	12
A	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9		NTC
B	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9	04.0002 pM Std	0.0002 pM Std
C	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9	0.002 pM Std	0.002 pM Std
D	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9	0.02 pM Std	0.02 pM Std
E	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9	0.2 pM Std	0.2 pM Std
F	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9	2 pM Std	2 pM Std
G	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9	20 pM Std	20 pM Std
H	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9

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	6	7	8	10	11	12
A	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9	NTC	NTC	NTC
B	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9	2 pM Std	2 pM Std	2 pM Std
G	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9	20 pM Std	20 pM Std	20 pM Std
H	ALF_PLT5_TN_16S_Col3	ALF_PLT5_TN_16S_Col9	ALF_PLT5_TN_ITS_Col3	ALF_PLT5_TN_ITS_Col9	ALF_PLT12_TN_16S_Col3	ALF_PLT12_TN_16S_Col9	ALF_PLT12_TN_ITS_Col3	ALF_PLT12_TN_ITS_Col9

Results:

Average results are below:

ALF_PLT5_TN_16S_Col3:

ALF_PLT5_TN_16S_Col9:

ALF_PLT5_TN_ITS_Col3:

ALF_PLT5_TN_ITS_Col9:

ALF_PLT12_TN_16S_Col3:

ALF_PLT12_TN_16S_Col9:

ALF_PLT12_TN_ITS_Col3:

ALF_PLT12_TN_ITS_Col9:

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.

100/Results = ul of Pool to Add

100/____ = ____ uL of Pool to Add

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

100- ____ = ____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.