Incubator vs Thermocycler comparison

We are expecting little to no difference. So, we will proceed with actual template, but the smaller PCR pool. We will compare via qPCR and bioanalyzer.

Restriction Digestion

(Keep MM and reaction plates on ice)

Add 3 ul Digestion MM to 22 plates using the 8 channel

Reagent	ul/rxn	rxns	ul needed
10x T4 Buffer	1.15	450	517.5
5M NaCl	0.12	450	54
1 mg/ml BSA	0.6	450	270
H2O	0.73	450	328.5
MseI (enzyme)	0.12	450	54
EcoR1 (enzyme)	0.28	450	126
Total	3	450	1350

Add 6 ul template to each plate with Benchsmart. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 65C for 1 hour. Do 1 set on 2 thermocyclers and 1 set in incubator (Somebody will have to return in 8 hours).

Adaptor Ligation

Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.

Reagent	ul/rxn	rxns	ul needed
MseI oligo	1	440	440
H2O	0.112	440	49.28
10x T4 Buffer	0.1	440	44
5M NaCl	0.01	440	4.4
1 mg/ml BSA	0.05	440	22
T4 DNA ligase	0.1675	440	73.7
Total	1.4	440	633.38

Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.

Template	EcoR1 MID plate
2017_ALFALFA_PLATE10	GBS Plate 5
2017_ALFALFA_PLATE11	GBS Plate 6

Label reaction plates with MID plate used.

Cover, vortex, and spin plates. Incubate thermocycler plate at 16C for 6 hours. Incubate incubator plate at RT for 2 hours.

Add 189 ul of low EDTA TE store at 4C for a month or -20C for longer.

PCR Amplification

Create the two pools below using 20uL from ligated plates matching well to well.

Pool	Plate1	Plate2	Plate3	Plate4
A	2017_ALFALFA_PLATE10_Thermocycler	2017_ALFALFA_PLATE11_Thermocycler
B	2017_ALFALFA_PLATE10_Incubator	2017_ALFALFA_PLATE11_Incubator

PCR1:

Reagent	ul/rxn	rxns	ul needed
H2O	9.52	220	2094.4
5x iProof buffer	4	220	880
10 mM dNTPs	0.4	220	88
50 mM MgCl2	0.4	220	88
5 uM Illumina Primers	1.33	220	292.6
iProof TAQ	0.2	220	44
DMSO	0.15	220	33
total	16	220	3520

Add 16 ul of PCR1 MM to each well of two new hard shell PCR plates.

Add 4uL of template from pooled plates.

Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:

Temp C	Cycles	Time
95*	1X	3:00
98	30X	0:30
60	30X	0:30
72	30X	0:40
72	1X	10:00
4*	1X*	0:00*

*pause step

Extra PCR

Add 2.125 ul of the MM directly to the previous PCR

Reagent	ul/rxn	rxns	ul needed
5x Iproof buffer	0.425	244	106.25
10 mM dNTPs	0.4	244	100
Primers	1.33	244	332.5
Total	2.155	244	538.75

Then continue with the last four unlisted steps for GBS1 from above:

Temp C	Cycles	Time
98	1X	3:00
60	1X	2:00
72	1X	10:00
4	1X	0:00

Comparisons

qPCR

Make 1:1000 dilutions of column 3 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:

	1	2	3	4	5	6	10	11	12
A	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12	NTC	NTC	NTC
B	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12	2 pM Std	2 pM Std	2 pM Std
G	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12	20 pM Std	20 pM Std	20 pM Std
H	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12

Combine 5uL from each well of GBS10-11_PCR_A into a pool. Make a 1:1000 dilution of this pool to run on qPCR.
Combine 5uL from each well of GBS10-11_PCR_B into a pool. Make a 1:1000 dilution of this pool to run on qPCR.
After 1:1000 pools have been made for each PCR plate, combine PCR_A and PCR_B undiluted pools together into one tube and label GBS10-11. Make a 1:1000 dilution of this pool to run on qPCR.
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)	90	900
2 ul	Primer Premix (10X)	90	180
4 ul	Ultra Pure Water	90	360
16 ul	Total Volume	90	1440

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	10	11	12
A	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12	GBS10-11 PCR_A Pool	NTC	NTC	NTC
B	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12	GBS10-11 PCR_A Pool	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12	GBS10-11 PCR_B Pool	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12	GBS10-11 PCR_B Pool	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12	GBS10-11 Pool	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12	GBS10-11 Pool	2 pM Std	2 pM Std	2 pM Std
G	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12		20 pM Std	20 pM Std	20 pM Std
H	GBS10-11 PCR_A Col_1	GBS10-11 PCR_A Col_6	GBS10-11 PCR_A Col_12	GBS10-11 PCR_B Col_1	GBS10-11 PCR_B Col_6	GBS10-11 PCR_B Col_12

Results:

Average quantities observed for all products:

GBS10-11 PCR_A Col_1:

GBS10-11 PCR_A Col_6:

GBS10-11 PCR_A Col_12:

GBS10-11 PCR_B Col_1:

GBS10-11 PCR_B Col_6:

GBS10-11 PCR_B Col_12:

GBS10-11 PCR_A Pool:

GBS10-11 PCR_B Pool:

GBS10-11 Pool:

GBS1-4 PCR_A Col_1:

GBS1-4 PCR_B Col_1:

Bioanalyzer

BioAnalyzer Chip Set up:

Well 1: GBS10-11 PCR_A Pool

Well 2: GBS10-11 PCR_B Pool

Well 3: GBS10-11 Pool

Well4: GBS10-11 PCR_A F3

Well 5: GBS10-11 PCR_A B5

Well 6: GBS10-11 PCR_A G7

Well 7: GBS10-11 PCR_A H12

Well 8: GBS10-11 PCR_B C1

Well 9: GBS10-11 PCR_B A4

Well 10: GBS10-11 PCR_B D6

Well 11: GBS10-11 PCR_B H8

Well 12: GBS10-11 PCR_B E11