Repeat PCR of RM_JAN22

RM_JAN22 is being repeated starting at PCR to increase the diversity of MIDs. Since we used two consecutive 16S MID plates for the first PCR, we suspect that this is causing the clusters to be too similar to properly pass through the flow cell. To fix this issue, the new PCR set up only used one MID plate (16S0H5) and was set up as follows:

	1	2	3	4	5	6	7	8	9	10	11	12

	1	2	3	4	5	8	9	10	11	12
A	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5
B	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5
C	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5
D	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5
E	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5
F	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5
G	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5
H	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5	RM_JAN22_Col1	RM_JAN22_Col2	RM_JAN22_Col3	RM_JAN22_Col4	RM_JAN22_Col5
	MID: 16S0H6 (Col1-Col5)					MID: 16S0H6 (Col8-Col12)

Sample Processing

Plate: RMJan22_II (repeat)

MasterMix

ul/rxn	Reagent	# of rxns	ul needed

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	80	300
0.45	10M dNTPs	80	45
0.3	Kapa HiFi HotStart DNA Pol	80	30
7.25	HPLC H2O	80	725
11	Total Volume	80	1100

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

Plate	16S MID

Plate	16S MID
RM_Jan22_II	16S0H6

Run on Thermocycler Program GSAF36:

Temp C	Cycles	Time

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 (16S Only):

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_MIDs)

qPCR

Make 1:1000 dilutions of all samples using a deep well plate.
Add 16 ul of Illumina Library Quantification MasterMix to each well:

ul/rxn	Reagent	# of rxns	ul needed

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	61	700
2 ul	Primer Premix (10X)	61	140
4 ul	Ultra Pure Water	61	280
16 ul	Total Volume	61	1120

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	10	11	12
A	RM_JAN22_II_16S_Col1	RM_JAN22_II_16S_Col2	RM_JAN22_II_16S_Col3	RM_JAN22_II_16S_Col4	RM_JAN22_II_16S_Col5	NTC	NTC	NTC
B	RM_JAN22_II_16S_Col1	RM_JAN22_II_16S_Col2	RM_JAN22_II_16S_Col3	RM_JAN22_II_16S_Col4	RM_JAN22_II_16S_Col5	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	RM_JAN22_II_16S_Col1	RM_JAN22_II_16S_Col2	RM_JAN22_II_16S_Col3	RM_JAN22_II_16S_Col4	RM_JAN22_II_16S_Col5	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	RM_JAN22_II_16S_Col1	RM_JAN22_II_16S_Col2	RM_JAN22_II_16S_Col3	RM_JAN22_II_16S_Col4	RM_JAN22_II_16S_Col5	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	RM_JAN22_II_16S_Col1	RM_JAN22_II_16S_Col2	RM_JAN22_II_16S_Col3	RM_JAN22_II_16S_Col4	RM_JAN22_II_16S_Col5	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	RM_JAN22_II_16S_Col1	RM_JAN22_II_16S_Col2	RM_JAN22_II_16S_Col3	RM_JAN22_II_16S_Col4	RM_JAN22_II_16S_Col5	2 pM Std	2 pM Std	2 pM Std
G	RM_JAN22_II_16S_Col1	RM_JAN22_II_16S_Col2	RM_JAN22_II_16S_Col3	RM_JAN22_II_16S_Col4	RM_JAN22_II_16S_Col5	20 pM Std	20 pM Std	20 pM Std
H	RM_JAN22_II_16S_Col1	RM_JAN22_II_16S_Col2	RM_JAN22_II_16S_Col3	RM_JAN22_II_16S_Col4	RM_JAN22_II_16S_Col5

Results:

Full result report can be viewed below:

Pool qPCR results:

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

1000/32.25 = 31uL of Pool to Add

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

1000- 31 = 969uL of 10mM Tris 8.5

*1nM pool will be combined with 1nM pool for the low read 2.5 experiment. Directions for iSeq prep found here.