4ul of Primers Amplicon MasterMix Prep and QC protocol_SH

*This is what I would do to QC for an entire library prep.

MasterMix (make for 84 plates in 2x 50mL conical tube)

Take out 13 tubes of dNTPs and 17 tubes of 5x Buffer to thaw.
Get six new 50 ml tubes from pre-PCR side and fill up with Ultra Pure Water from the EcoBGC. Label tubes 1-6.
Using the 25mL combitip, aliquot 22,648.5uL into a new pre-PCR side 50mL tube. Repeat again into a second new pre-PCR side 50mL tube.
Once thawed, combine all 17 tubes of 5x Buffer into a 50mL tube labelled 5x KAPA Hifi Buffer
Once thawed, combine all 13 tubes of dNTPs into a 5mL tube labelled 10mM dNTP
Combine 11 tubes of KAPA Hifi HS DNA Pol into one 5mL tube labelled KAPA Hifi HS DNA Pol
Add the remaining master mix reagents to each tube in the quantities listed below using appropriate combitips or pipettes.

ul/rxn	Reagent	# of rxns	ul needed

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	4314	12942
0.45	10mM dNTPs	4314	1941.3
0.3	Kapa HiFi HotStart DNA Pol	4314	1294.2
5.25	HPLC H2O	4314	22648.5
11	Total Volume	4314	47454

Aliquot 4500 ul into ten 5ml tubes and the remaining 2,454uL into an additional 5mL tube. Label AmpStd_MMX1_Prep-Date*_NS#** and AmpStd_MMX2_Prep-Date_NS#.

*Prep-Date is the day the mastermix is prepped, i.e. 06/17/22

**NS# = NS7 or whatever Novaseq run it may be affiliated with

Aliquot 9 ul into each well of four pcr-tube strip using a 1mL combitip or regular pipette. Add the following to the wells below:

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

	1	2	3	4	5	6	10	11	12
A	MMX1 + 2uL 16SF+ 2ul 16SR + 2uL TE Bottle 1	MMX1 + 2uL 16SF+ 2ul 16SR +2uL TE Bottle 1	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL TE Bottle 1	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL TE Bottle 1	MMX2 + 2uL ILLP + 4uL TE Bottle 1	MMX2 + 2uL ILLP + 4uL TE Bottle 1	NTC	NTC	NTC
B	MMX1 + 2uL 16SF+ 2ul 16SR + 2uL TE Bottle 2	MMX1 + 2uL 16SF+ 2ul 16SR + 2uL TE Bottle 2	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL TE Bottle 2	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL TE Bottle 2	MMX2 + 2uL ILLP + 4uL TE Bottle 2	MMX2 + 2uL ILLP + 4uL TE Bottle 2	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	MMX1 + 2uL 16SF+ 2ul 16SR + 2uL UPW 3	MMX1 + 2uL 16SF+ 2ul 16SR + 2uL UPW 3	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL UPW 3	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL UPW 3	MMX2 + 2uL ILLP + 4uL UPW 3	MMX2 + 2uL ILLP + 4uL UPW 3	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	MMX1 + 2uL 16SF+ 2ul 16SR + 2uL UPW 4	MMX1 + 2uL 16SF+ 2ul 16SR + 2uL UPW 4	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL UPW 4	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL UPW 4	MMX2 + 2uL ILLP + 4uL UPW 4	MMX2 + 2uL ILLP + 4uL UPW 4	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	MMX1 + 2uL 16SF+ 2ul 16SR + 2uL UPW 5	MMX1 + 2uL 16SF+ 2ul 16SR + 2uL UPW 5	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL UPW 5	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL UPW 5	MMX2 + 2uL ILLP + 4uL UPW 5	MMX2 + 2uL ILLP + 4uL UPW 5	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	MMX1 + 2uL 16SF+ 2ul 16SR + 2uL UPW 6	MMX1 + 2uL 16SF+ 2ul 16SR + 2uL UPW 6	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL UPW6	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL UPW6	MMX2 + 2uL ILLP + 4uL UPW 6	MMX2 + 2uL ILLP + 4uL UPW 6	2 pM Std	2 pM Std	2 pM Std
G	MMX1 + 2uL 16SF+ 2ul 16SR + 2uL MC	MMX1 + 2uL 16SF+ 2ul 16SR + 2uL MC	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL MC	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL MC	MMX2 + 2uL ILLP + 4uL MC	MMX2 + 2uL ILLP + 4uL MC	20 pM Std	20 pM Std	20 pM Std
H	MMX1 + 2uL 16SF+ 2ul 16SR + 2uL MC	MMX1 + 2uL 16SF+ 2ul 16SR + 2uL MC	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL MC	MMX1 + 2uL ITSF+ 2ul ITSR + 2uL MC	MMX2 + 2uL ILLP + 4uL MC	MMX2 + 2uL ILLP + 4uL MC

MMX2 NOTE: Master-mix should not go through more than three freeze-thaws. If using a reservoir, pour any leftover mastermix into a new labelled tube and mark with a tally for freeze-thaws if mastermix was previously frozen. If using a combitip, close mastermix tube and mark with a tally to keep track of freeze thaws. If mastermix is being QC’d immediately after being made, no tally is needed until the master mix has been frozen.

Cap, Vortex, and spin down. 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 Columns 1, 3, & 5:

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)
Store in -20 until ready to qPCR.

qPCR MMX & Reagent QC

Make 1:1000 dilutions of column 8 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	9	10	11	12

	1	2	3	4	5	6	10	11	12
A	MMX1_16S_TE1_Clean	MMX1_16S_TE1	MMX1_ITS_TE1_Clean	MMX1_ITS_TE1	MMX2_ILLP_TE1_Clean	MMX2_ILLP_TE1	NTC	NTC	NTC
B	MMX1_16S_TE2_Clean	MMX1_16S_TE2	MMX1_ITS_TE2_Clean	MMX1_ITS_TE2	MMX2_ILLP_TE2_Clean	MMX2_ILLP_TE2	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	MMX1_16S_UPW3_Clean	MMX1_16S_UPW3	MMX1_ITS_UPW3_Clean	MMX1_ITS_UPW3	MMX2_ILLP_UPW3_Clean	MMX2_ILLP_UPW3	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	MMX1_16S_UPW4_Clean	MMX1_16S_UPW4	MMX1_ITS_UPW4_Clean	MMX1_ITS_UPW4	MMX2_ILLP_UPW4_Clean	MMX2_ILLP_UPW4	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	MMX1_16S_UPW5_Clean	MMX1_16S_UPW5	MMX1_ITS_UPW5_Clean	MMX1_ITS_UPW5	MMX2_ILLP_UPW5_Clean	MMX2_ILLP_UPW5	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	MMX1_16S_UPW6_Clean	MMX1_16S_UPW6	MMX1_ITS_UPW6_Clean	MMX1_ITS_UPW6	MMX2_ILLP_UPW6_Clean	MMX2_ILLP_UPW6	2 pM Std	2 pM Std	2 pM Std
G	MMX1_16S_MC_Clean	MMX1_16S_MC	MMX1_ITS_MC_Clean	MMX1_ITS_MC	MMX2_ILLP_MC_Clean	MMX2_ILLP_MC	20 pM Std	20 pM Std	20 pM Std
H	MMX1_16S_MC_Clean	MMX1_16S_MC	MMX1_ITS_MC_Clean	MMX1_ITS_MC	MMX2_ILLP_MC_Clean	MMX2_ILLP_MC

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)	80	800
2 ul	Primer Premix (10X)	80	160
4 ul	Ultra Pure Water	80	240
16 ul	Total Volume	80	1200

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	10	11	12
A	MMX1_16S_TE1_Clean	MMX1_16S_TE1	MMX1_ITS_TE1_Clean	MMX1_ITS_TE1	MMX2_ILLP_TE1_Clean	MMX2_ILLP_TE1	20 pM Std	20 pM Std	20 pM Std
B	MMX1_16S_TE2_Clean	MMX1_16S_TE2	MMX1_ITS_TE2_Clean	MMX1_ITS_TE2	MMX2_ILLP_TE2_Clean	MMX2_ILLP_TE2	2 pM Std	2 pM Std	2 pM Std
C	MMX1_16S_UPW3_Clean	MMX1_16S_UPW3	MMX1_ITS_UPW3_Clean	MMX1_ITS_UPW3	MMX2_ILLP_UPW3_Clean	MMX2_ILLP_UPW3	0.2 pM Std	0.2 pM Std	0.2 pM Std
D	MMX1_16S_UPW4_Clean	MMX1_16S_UPW4	MMX1_ITS_UPW4_Clean	MMX1_ITS_UPW4	MMX2_ILLP_UPW4_Clean	MMX2_ILLP_UPW4	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	MMX1_16S_UPW5_Clean	MMX1_16S_UPW5	MMX1_ITS_UPW5_Clean	MMX1_ITS_UPW5	MMX2_ILLP_UPW5_Clean	MMX2_ILLP_UPW5	0.002 pM Std	0.002 pM Std	0.002 pM Std
F	MMX1_16S_UPW6_Clean	MMX1_16S_UPW6	MMX1_ITS_UPW6_Clean	MMX1_ITS_UPW6	MMX2_ILLP_UPW6_Clean	MMX2_ILLP_UPW6	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
G	MMX1_16S_MC_Clean	MMX1_16S_MC	MMX1_ITS_MC_Clean	MMX1_ITS_MC	MMX2_ILLP_MC_Clean	MMX2_ILLP_MC	NTC	NTC	NTC
H	MMX1_16S_MC_Clean	MMX1_16S_MC	MMX1_ITS_MC_Clean	MMX1_ITS_MC	MMX2_ILLP_MC_Clean	MMX2_ILLP_MC	NTC	NTC	NTC

Results:

Plate Name	Average Result (nanomoles)

Plate Name	Average Result (nanomoles)

Full qPCR results below:

Reagent	QC Passed Y/N

Reagent	QC Passed Y/N
MMX1
MMX2
TE Bottle 1 Lot:
TE Bottle 2 Lot:
UPW 1
UPW 2
UPW 3
UPW 4
UPW 5
UPW 6
MagBead Lot:

If QC passes, aliquot all remaining UPW into 2mL tubes and label with UPW# and aliquot date.
If QC passes, aliquot TE bottles into a variety of 5mL, 15mL, and 50mL tubes and label with bottle number, lot number, and aliquot date.
You could aliquot the MagBeads like the TE as well unless it is not advised to aliquot out that reagent. TBD.

NOTE: You can scale this back and only QC TE, UPW, and MagBeads just using the first MMX instead of both. You can just QC MMX2 using TE or one UPW tube as the sample, and skip the MagBead for MMX2 as well and just MagBead 1 & 3. This would allow you to run just two columns for MMX2 for 16S and ITS, allowing for 6 lanes instead of 8. I honestly would probably do that instead but just in case you were wanting to look at comparability data between master mixes I just made them the same.