Page Comparison

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

PCR Amplification

Pool Create the two pools below using 20uL from ligated plates matching well to well in sets of 4 with one set of 2 avoiding combining the same MIDs together:.

Pool	Plate1	Plate2	Plate3	Plate4
6aA	2017_ALFALFA_PLATE10_Thermocycler	2017_ALFALFA_PLATE11_Thermocycler
6bB	2017_ALFALFA_PLATE10_Incubator	2017_ALFALFA_PLATE11

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_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:

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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

Versions Compared

Old Version 5

New Version 6

Key

PCR Amplification

PCR1:

Comparisons

qPCR

Results:

Bioanalyzer

Bioanalyzer