snplex genotyping system, 48-plex€¦ · quick reference card snplex™ genotyping system, 48-plex...
TRANSCRIPT
SNPlex™ Genotyping System, 48-plex
SNPlex™ Genotyping System, 48-plexQuick Reference Card
For safety and biohazard guidelines, refer to the “Safety” section in the SNPlex™ Genotyping System 48-plex User Guide (PN 4360856). For all chemicals in red bold type below, read the MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.
This quick reference card provides simplified procedures for using the SNPlex Genotyping System with the Applied Biosystems 3730/3730xl DNA Analyzer and 3130xl Genetic Analyzer.
Applied Biosystems recommends using only the plate covers listed in Table 1 in “SNPlex Genotyping System, 48-plex Required Equipment and Consumables” on page 14. (This table also appears in the SNPlex™ Genotyping System 48-plex User Guide on page 1-11.)
For detailed procedures and ordering information, see the SNPlex™ Genotyping System 48-plex User Guide.
SNPlex™ System WorkflowTwo laboratories are required. The following illustration summarizes the division of procedures between the two laboratories.
Prepare gDNA
Design sample plate layout
Design and orderSNPlex™ ligation probes
Purify OLA products(Exonuclease)
Phosphorylate andligate probes andlinkers to gDNA
(OLA wet)
OLA
Lab
orat
ory
Run PCR
Prepare hybridization platesand bind PCR product
to plates
Add denaturant, isolatingbiotinylated strand on
hybridization plate
Prepare sample platesfor electrophoresis
Create results groupsand plate records
Load and runsample plates
Analyze data inGeneMapper® software v4.0
Elute ZipChute probes
Hybridize ZipChute® probes
PC
R L
abor
ator
y
Prepare PCR reactions
Phosphorylate andligate probes and linkers to gDNA
(OLA dry)
Dilute purified OLA products
Am
plifi
catio
nK
it
Ass
ay S
tand
ards
Kit
Pur
ifica
tion
Kit
Olig
onuc
leot
ide
Liga
tion
Kit
Hyb
ridiz
atio
n R
eage
nts
and
Kits
QUICK REFERENCE CARD
Setting Up the Applied Biosystems 3730/3730xl and 3130xl DNA Analyzers for SNPlex System Experiments
Supported Configuration
The protocols described in this QRC are developed for 3730/3730xl and 3130xl instruments running Data Collection Software v2.0 or higher and GeneMapper® Software v4.0 or higher.
Checklist
Sample Plate Layout ExamplesThe table on pages 3 and 4 illustrates examples of sample layouts for 384-well and 96-well plates. The setups assume that there are four probe pools per 384-well plate and one probe pool per 96-well plate. The number of gDNA samples, controls, NTCs, and allelic ladders differs between 96-capillary, 48-capillary, and 16-capillary instruments.
Task Description
Have you installed the files required by the Data Collection Software?a
a These instructions apply to Data Collection v2.0 only. Upon upgrading to Data Collection software v3.0, the module (HTSNP36_POP7_V3), dye set, and prebatch file are installed automatically.
Install Prebatch.txt, HTSNP36_POP7_V2.xml, and Dye Set S (S.zip). The files are available on the SNPlex™ System 48-plex Support Files CD (PN 4352129).
Have you created a SNPlex System Instrument Protocol?
Create an instrument protocol that associates the appropriate run module with Dye Set S:
• HTSNP36_POP7_V2• HTSNP36_POP7_V3• HTSNP50_POP7
Have you upgraded GeneMapper software to v4.0 and installed the parameter files required by the GeneMapper Software?
Upgrade the GeneMapper software and install the parameter files as explained in the GeneMapper® Software v4.0 Installation Guide (PN 4363080). The files are available on the GeneMapper software v4.0 installation CD or as a download from www.appliedbiosystems.com.
Have you preconditioned the capillary array?b
b Preconditioning applies to new arrays only.
Fill the array with diluted Array Conditioning Buffer, incubate for 30 min, then rinse with deionized water before installing the array on the instrument.
Have you performed spatial and spectral calibrations?
Refer to the SNPlex™ Genotyping System 48-plex User Guide for information about performing spatial and spectral calibrations.
Have you validated instrument performance? To assess the signal intensity and resolution, perform a mock run using a diluted solution of the SNPlex™ ZipChute™ Mix and internal size standard, as explained in the SNPlex™ Genotyping System 48-plex User Guide.
Page 2
384-wells, 96-capillary array 96-wells, 96-capillary array
384-wells, 48-capillary array 96-wells, 48-capillary array
P
O
N
M
L
K
J
I
H
G
F
E
D
C
B
A1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
P
O
N
M
L
K
J
I
H
G
F
E
D
C
B
A1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
P
O
N
M
L
K
J
I
H
G
F
E
D
C
B
A1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
P
O
N
M
L
K
J
I
H
G
F
E
D
C
B
A1
Injection 1 Injection 2
Injection 3 Injection 4
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
89
90
91
92
C
N
L
L
81
82
83
84
85
86
87
88
73
74
75
76
77
78
79
80
65
66
67
68
69
70
71
72
57
58
59
60
61
62
63
64
49
50
51
52
53
54
55
56
41
42
43
44
45
46
47
48
33
34
35
36
37
38
39
40
25
26
27
22
29
30
31
32
17
18
19
20
21
22
23
24
9
10
11
12
13
14
15
16
1
C
N
L
L
2
3
4
5
6
7
8
C
N
L
L
8981736557494133251791
90827466585042342618102
91837567595143352719113
92847668605244362820124
857769615345372921135
867870625446383022146
877971635547393123157
888072645648403224168
1
1
2
3
4
5
6
7
8
2
3
4
5
6
7
8
9
9
10
11
12
13
14
15
16
10
11
12
13
14
15
16
17
17
18
19
20
21
22
23
24
18
19
20
21
22
23
24
25
25
26
27
28
29
30
31
32
26
27
28
29
30
31
32
33
33
34
35
36
37
38
39
40
34
35
36
37
38
39
40
41
41
42
43
44
45
46
47
48
42
43
44
45
46
47
48
49
49
50
51
52
53
54
55
56
50
51
52
53
54
55
56
57
57
58
59
60
61
62
63
64
58
59
60
61
62
63
64
65
65
66
67
68
69
70
71
72
66
67
68
69
70
71
72
73
73
74
75
76
77
78
79
80
74
75
76
77
78
79
80
81
81
82
83
84
85
86
87
88
82
83
84
85
86
87
88
89
89
90
91
92
90
91
92
C
N
L
L
C
N
L
L
8981736557494133251791
90827466585042342618102
91837567595143352719113
92847668605244362820124
C857769615345372921135
N867870625446383022146
L877971635547393123157
L888072645648403224168
C
N
L
L
1
1
2
3
4
5
6
7
8
2
3
4
5
6
7
8
9
9
10
11
12
13
14
15
16
10
11
12
13
14
15
16
17
17
18
19
20
21
22
23
24
18
19
20
21
22
23
24
25
25
26
27
28
29
30
31
32
26
27
28
29
30
31
32
33
33
34
35
36
37
38
39
40
34
35
36
37
38
39
40
41
41
42
43
44
45
46
47
48
42
43
44
45
46
47
48
49
49
50
51
52
53
54
55
56
50
51
52
53
54
55
56
57
57
58
59
60
61
62
63
64
58
59
60
61
62
63
64
65
65
66
67
68
69
70
71
72
66
67
68
69
70
71
72
73
73
74
75
76
77
78
79
80
74
75
76
77
78
79
80
81
81
82
83
84
85
86
87
88
82
83
84
85
86
87
88
89
90
91
92
81
82
83
84
85
86
87
88
73
74
75
76
77
78
79
80
65
66
67
68
69
70
71
72
57
58
59
60
61
62
63
64
49
50
51
52
53
54
55
56
41
42
43
44
45
46
47
48
33
34
35
36
37
38
39
40
25
26
27
22
29
30
31
32
17
18
19
20
21
22
23
24
9
10
11
12
13
14
15
16
1
2
3
4
5
6
7
8
89
89
90
91
92
90
91
92
C
N
L
L
C
N
L
L
C
N
L
L
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
C
N
L
L
89
90
91
92
81
82
83
84
85
86
87
88
73
74
75
76
77
78
79
80
65
66
67
68
69
70
71
72
57
58
59
60
61
62
63
64
49
50
51
52
53
54
55
56
41
42
43
44
45
46
47
48
33
34
35
36
37
38
39
40
25
26
27
22
29
30
31
32
17
18
19
20
21
22
23
24
9
10
11
12
13
14
15
16
1
8981736557494133251791
90827466585042342618102
91837567595143352719113
92847668605244362820124
857769615345372921135
867870625446383022146
877971635547393123157
888072645648403224168
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
C
N
L
L
C
N
L
L
C
N
L
L
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
82
83
84
85
86
87
88
89
89
90
91
92
81
82
83
84
85
86
87
88
73
74
75
76
77
78
79
80
65
66
67
68
69
70
71
72
57
58
59
60
61
62
63
64
49
50
51
52
53
54
55
56
41
42
43
44
45
46
47
48
33
34
35
36
37
38
39
40
25
26
27
22
29
30
31
32
17
18
19
20
21
22
23
24
9
10
11
12
13
14
15
16
1
8981736557494133251791
90827466585042342618102
91837567595143352719113
92847668605244362820124
857769615345372921135
867870625446383022146
877971635547393123157
888072645648403224168
2
3
4
5
6
7
8
89
90
91
92
C
N
L
L
90
91
92
C
N
L
L
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
85 C
86 N
87 L
1 17 33 49 65 81
2 18 34 50 66 82
3 19 35 51 67 83
4 20 36 52 68 84
5 21 37 53 69
6 22 38 54 70
7 23 39 55 71
8 24 40 56 72
9 25 41 57 73 89
10 26 42 58 74 90
11 27 43 59 75 91
12 28 44 60 76 92
13 29 45 61 77
14 30 46 62 78
15 31 47 63 79
16 32 48 64 80 88 L
Yellow
Green
= Probe Set A, Quadrant 1
= Probe Set B, Quadrant 2
1 to 92 = gDNA samples
C = Control DNA
Blue
Orange
= Probe Set C, Quadrant 3
= Probe Set D, Quadrant 4
N = NTC
L = Allelic Ladder
P
O
N
M
L
K
J
I
H
G
F
E
D
C
B
A1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Injection 1
P
O
N
M
L
K
J
I
H
G
F
E
D
C
B
A1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
P
O
N
M
L
K
J
I
H
G
F
E
D
C
B
A1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
P
O
N
M
L
K
J
I
H
G
F
E
D
C
B
A1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Injection 3
Injection 2
Injection 4
Injection 5
Injection 7
Injection 6
Injection 8
C
N
L
L
81
82
83
84
65
66
67
68
69
70
71
72
49
50
51
52
53
54
55
56
33
34
35
36
37
38
39
40
17
18
19
20
21
22
23
24
1
C
N
L
L
2
3
4
5
6
7
8
C
N
L
L
81654933171
82665034182
83675135193
84685236204
695337215
705438226
715539237
725640248
85
86
87
88
73
74
75
76
77
78
79
80
57
58
59
60
61
62
63
64
41
42
43
44
45
46
47
48
25
26
27
22
29
30
31
32
9
10
11
12
13
14
15
16
85735741259
867458422610
877559432711
887660442812
7761452913
7862463014
7963473115
8064483216
1
1
2
3
4
5
6
7
8
2
3
4
5
6
7
8
9
9
10
11
12
13
14
15
16
10
11
12
13
14
15
16
17
17
18
19
20
21
22
23
24
18
19
20
21
22
23
24
25
25
26
27
28
29
30
31
32
26
27
28
29
30
31
32
33
33
34
35
36
37
38
39
40
34
35
36
37
38
39
40
41
41
42
43
44
45
46
47
48
42
43
44
45
46
47
48
49
49
50
51
52
53
54
55
56
50
51
52
53
54
55
56
57
57
58
59
60
61
62
63
64
58
59
60
61
62
63
64
65
65
66
67
68
69
70
71
72
66
67
68
69
70
71
72
73
73
74
75
76
77
78
79
80
74
75
76
77
78
79
80
81
81
82
83
84
82
83
84
85
85
86
87
88
86
87
88
C
N
L
L
C
N
L
L
C
N
L
L
C
N
L
L
C
N
L
L
85
86
87
88
C
N
L
L
73
74
75
76
77
78
79
80
57
58
59
60
61
62
63
64
41
42
43
44
45
46
47
48
25
26
27
22
29
30
31
32
9
10
11
12
13
14
15
16
C
N
L
L
C
N
L
L
85735741259
867458422610
877559432711
887660442812
7761452913
7862463014
7963473115
8064483216
81
82
83
84
65
66
67
68
69
70
71
72
49
50
51
52
53
54
55
56
33
34
35
36
37
38
39
40
17
18
19
20
21
22
23
24
1
81654933171
82665034182
83675135193
84685236204
695337215
705438226
715539237
725640248
1
1
2
3
4
5
6
7
8
2
3
4
5
6
7
8
2
3
4
5
6
7
8
9
9
10
11
12
13
14
15
16
10
11
12
13
14
15
16
17
17
18
19
20
21
22
23
24
18
19
20
21
22
23
24
25
25
26
27
28
29
30
31
32
26
27
28
29
30
31
32
33
33
34
35
36
37
38
39
40
34
35
36
37
38
39
40
41
41
42
43
44
45
46
47
48
42
43
44
45
46
47
48
49
49
50
51
52
53
54
55
56
50
51
52
53
54
55
56
57
57
58
59
60
61
62
63
64
58
59
60
61
62
63
64
65
65
66
67
68
69
70
71
72
66
67
68
69
70
71
72
73
73
74
75
76
77
78
79
80
74
75
76
77
78
79
80
81
81
82
83
84
82
83
84
85
85
86
87
88
86
87
88
C
N
L
L
C
N
L
L
C
N
L
L
C
N
L
L
C
N
L
L
C
N
L
L
81654933171
82665034182
83675135193
84685236204
C695337215
N705438226
L715539237
L725640248
81
82
83
84
65
66
67
68
69
70
71
72
49
50
51
52
53
54
55
56
33
34
35
36
37
38
39
40
17
18
19
20
21
22
23
24
1
2
3
4
5
6
7
8
C
N
L
L
85
86
87
88
73
74
75
76
77
78
79
80
57
58
59
60
61
62
63
64
41
42
43
44
45
46
47
48
25
26
27
22
29
30
31
32
9
10
11
12
13
14
15
16
85735741259
867458422610
877559432711
887660442812
7761452913
7862463014
7963473115
8064483216
1
1
2
3
4
5
6
7
8
2
3
4
5
6
7
8
9
9
10
11
12
13
14
15
16
10
11
12
13
14
15
16
17
17
18
19
20
21
22
23
24
18
19
20
21
22
23
24
25
25
26
27
28
29
30
31
32
26
27
28
29
30
31
32
33
33
34
35
36
37
38
39
40
34
35
36
37
38
39
40
41
41
42
43
44
45
46
47
48
42
43
44
45
46
47
48
49
49
50
51
52
53
54
55
56
50
51
52
53
54
55
56
57
57
58
59
60
61
62
63
64
58
59
60
61
62
63
64
65
65
66
67
68
69
70
71
72
66
67
68
69
70
71
72
73
73
74
75
76
77
78
79
80
74
75
76
77
78
79
80
81
81
82
83
84
82
83
84
85
85
86
87
88
86
87
88
C
N
L
L
C
N
L
L
C
N
L
L
C
N
L
L
C
N
L
L
C
N
L
L
85
86
87
88
73
74
75
76
77
78
79
80
57
58
59
60
61
62
63
64
41
42
43
44
45
46
47
48
25
26
27
22
29
30
31
32
9
10
11
12
13
14
15
16
85735741259
867458422610
877559432711
887660442812
C7761452913
N7862463014
L7963473115
L8064483216
C
N
L
L
81
82
83
84
65
66
67
68
69
70
71
72
49
50
51
52
53
54
55
56
33
34
35
36
37
38
39
40
17
18
19
20
21
22
23
24
1
81654933171
82665034182
83675135193
84685236204
695337215
705438226
715539237
725640248
1
1
2
3
4
5
6
7
8
2
3
4
5
6
7
8
2
3
4
5
6
7
8
9
9
10
11
12
13
14
15
16
10
11
12
13
14
15
16
17
17
18
19
20
21
22
23
24
18
19
20
21
22
23
24
25
25
26
27
28
29
30
31
32
26
27
28
29
30
31
32
33
33
34
35
36
37
38
39
40
34
35
36
37
38
39
40
41
41
42
43
44
45
46
47
48
42
43
44
45
46
47
48
49
49
50
51
52
53
54
55
56
50
51
52
53
54
55
56
57
57
58
59
60
61
62
63
64
58
59
60
61
62
63
64
65
65
66
67
68
69
70
71
72
66
67
68
69
70
71
72
73
73
74
75
76
77
78
79
80
74
75
76
77
78
79
80
81
81
82
83
84
82
83
84
85
85
86
87
88
86
87
88
C
N
L
L
C
N
L
L
C
N
L
L
C
N
L
L
C
N
L
L
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
C C
N N
L L
1 17 33 49 65 81
2 18 34 50 66 82
3 19 35 51 67 83
4 20 36 52 68 84
5 21 37 53 69
6 22 38 54 70
7 23 39 55 71
8 24 40 56 72
9 25 41 57 73 85
10 26 42 58 74 86
11 27 43 59 75 87
12 28 44 60 76 88
13 29 45 61 77
14 30 46 62 78
15 31 47 63 79
16 32 48 64 80 L L
Injection 1
1 to 88 = gDNA samples C = Control DNA
N = NTC L = Allelic LadderA
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
C C
N N
L L
1 17 33 49 65 81
2 18 34 50 66 82
3 19 35 51 67 83
4 20 36 52 68 84
5 21 37 53 69
6 22 38 54 70
7 23 39 55 71
8 24 40 56 72
9 25 41 57 73 85
10 26 42 58 74 86
11 27 43 59 75 87
12 28 44 60 76 88
13 29 45 61 77
14 30 46 62 78
15 31 47 63 79
16 32 48 64 80 L L
Injection 2
Yellow
Green
= Probe Set A, Quadrant 1
= Probe Set B, Quadrant 2
1 to 88 = gDNA samples
C = Control DNA
Blue
Orange
= Probe Set C, Quadrant 3
= Probe Set D, Quadrant 4
N = NTC
L = Allelic Ladder
Page 3
384-wells, 16-capillary array
96-wells, 16-capillary array
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
80 88
81 N
82 C
1 16 31 46 61 76
2 17 32 47 62 77
3 18 33 48 63 78
4 19 34 49 64 79
5 20 35 50 65
6 21 36 51 66
7 22 37 52 67
8 23 38 53 68
9 24 39 54 69 84
10 25 40 55 70 85
11 26 41 56 71 86
12 27 42 57 72 87
13 28 43 58 73
14 29 44 59 74
15 30 45 60 75
L L L L L 83 L
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
1 16 31 46 61
2 17 32 47 62
3 18 33 48 63
4 19 34 49 64
5 20 35 50 65
6 21 36 51 66
7 22 37 52 67
8 23 38 53
9 24 39 54
10 25 40 55
11 26 41 56
12 27 42 57
13 28 43 58
14 29 44 59
15 30 45 60
L L L L L L
Injection 1
Injection 2
80 88
81 N
82 C
76
77
78
79
68
69 84
70 85
71 86
72 87
73
74
75
83
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
1 16 31 46 61
2 17 32 47 62
3 18 33 48 63
4 19 34 49 64
5 20 35 50 65
6 21 36 51 66
7 22 37 52 67
8 23 38 53
9 24 39 54
10 25 40 55
11 26 41 56
12 27 42 57
13 28 43 58
14 29 44 59
15 30 45 60
L L L L L L
Injection 3
80 88
81 N
82 C
76
77
78
79
68
69 84
70 85
71 86
72 87
73
74
75
83
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
1 16 31 46 61
2 17 32 47 62
3 18 33 48 63
4 19 34 49 64
5 20 35 50 65
6 21 36 51 66
7 22 37 52 67
8 23 38 53
9 24 39 54
10 25 40 55
11 26 41 56
12 27 42 57
13 28 43 58
14 29 44 59
15 30 45 60
L L L L L L
Injection 4
80 88
81 N
82 C
76
77
78
79
68
69 84
70 85
71 86
72 87
73
74
75
83
Injection 5
Injection 6
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
1 16 31 46 61
2 17 32 47 62
3 18 33 48 63
4 19 34 49 64
5 20 35 50 65
6 21 36 51 66
7 22 37 52 67
8 23 38 53
9 24 39 54
10 25 40 55
11 26 41 56
12 27 42 57
13 28 43 58
14 29 44 59
15 30 45 60
L L L L L L
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
1 16 31 46 61
2 17 32 47 62
3 18 33 48 63
4 19 34 49 64
5 20 35 50 65
6 21 36 51 66
7 22 37 52 67
8 23 38 53
9 24 39 54
10 25 40 55
11 26 41 56
12 27 42 57
13 28 43 58
14 29 44 59
15 30 45 60
L L L L L L
80 88
81 N
82 C
76
77
78
79
68
69 84
70 85
71 86
72 87
73
74
75
83
80 88
81 N
82 C
76
77
78
79
68
69 84
70 85
71 86
72 87
73
74
75
83
1 to 88 = gDNA samplesC = control DNAN = NTCL = Allelic Ladder
Page 4
Reagent WorksheetApplied Biosystems recommends copying this worksheet and using it to calculate the volumes you need for each step in the assay.
Assay Step Description Volume Calculation Total Volume Lot No.
Phosphorylation and Ligation
OLA Master Mix 2.50 µL × number of samples × µL overage
Nuclease-free water + dried-down DNAorNuclease-free water + wet DNA
2.30 µL × number of samples × µL overage
0.30 µL × number of samples × µL overage
Universal linkers 0.05 µL × number of samples × µL overage
dATP 0.05 µL × number of samples × µL overage
SNPlex™ ligation probes 0.1 µL × number of samples × µL overage
Purification
Nuclease-free water 4.2 µL × number of samples × µL overage
Exonuclease buffer (10✕) 0.5 µL × number of samples × µL overage
Lambda exonuclease 0.2 µL × number of samples × µL overage
Exonuclease 0.1 µL × number of samples × µL overage
PCR
Nuclease-free water 2.4 µL × number of samples × µL overage
Amplification master mix 5 µL × number of samples × µL overage
Amplification primers 0.5 µL × number of samples × µL overage
Diluted Wash Buffer
Nuclease-free water 90 µL × number of samples × µL overage × 7a
a Number of total wash steps.
Wash buffer (10✕) 10 µL × number of samples × µL overage × 7a
PCR Binding
Binding buffer 17.491 µL × number of samples × µL overage
Positive hybridization control 0.009 µL × number of samples × µL overage
ZipChute™ Probe Hybridization
ZipChute probe mix 0.05 µL × number of samples × µL overage
Denaturant 11.25 µL × number of samples × µL overage
ZipChute dilution buffer 13.7 µL × number of samples × µL overage
Sample Loading PreparationSize standard 0.6 µL × number of samples × µL overage
Sample loading reagent 16.9 µL × number of samples × µL overage
Allelic Ladder Dispense96-well plates 1.25 µL
384-well plates 1.0 µL
Page 5
SNPlex Genotyping System 48-plex ProtocolThis protocol provides instructions for manually performing SNPlex™ System experiments using 96- and 384-well plates. All volumes are calculated for single reactions and need to be scaled-up appropriately. The SNPlex™ Genotyping System 48-plex General Automation Getting Started Guide (PN 4358099) provides modified protocols for automating the SNPlex System assay using robotics.
1 Prepare and fragment gDNA. a. Purify gDNA using one of the recommended kits. (Refer to the SNPlex™ Genotyping System 48-plex User Guide for a list of recommended kits.)
b. Determine the concentration of gDNA. Optionally, run an aliquot of each quantified DNA sample on a 0.8% agarose gel. If the sample appears as a solid, high-molecular-weight band, continue with the procedure. If the sample appears smeared across the lane, omit the heat-fragmentation step.Note: Heat fragmentation has been shown to improve genotyping of some SNPs. However, over-fragmentation is detrimental to the assay. Heat fragmentation may be omitted if desired. For DNA samples available only in limited quantities, it is recommended you omit heat fragmentation and proceed to step g.Note: Applied Biosystems recommends using the TaqMan® RNase P Quantification Kit to quantify the concentration of human DNA. Other methods can result in false-high calculations of gDNA concentration, which may negatively affect the assay.
c. Dilute the purified DNA to a concentration of 50 to 200 ng/µL using 1✕ TE, pH 8.0. Note: The fragmentation protocol is equally effective if the DNA sample is diluted in nuclease-free water, 0.5✕ TE pH 8.0, 2✕ TE pH 8.0, 1✕ TE pH 7.5, 1✕ TE pH 7.0, Gentra’s PureGene® DNA Hydration Solution, or Qiagen’s FlexiGene Hydration Buffer.
d. Dispense 12.5 to 150 µL of prepared gDNA into a chilled reaction plate, then cover the plate.
e. Program the thermal cycler as follows:
f. When the thermal cycler reaches 4 ° C, insert the chilled reaction plate and resume the program.
g. Dilute the fragmented gDNA to a final concentration of 18.5 ng/µL with 1✕ TE, pH 8.0.
Note: If using whole genome amplification (WGA), Applied Biosystems recommends diluting the DNA to 37 ng/µL.
Note: The concentration of 18.5 ng/µL is based on quantification using the TaqMan® RNase P Quantification Assay. If you are using fluorescence- or absorbance-based assays, consider doubling the gDNA concentration (37 ng/µL).
h. Dispense 2 µL of diluted DNA into each well of a 384- or 96-well optical reaction plate. Air-dry for 3 days in a dark, amplicon-free location. After drying, keep the plate covered in the dark at room temperature until use (dried-down gDNA method).Alternatively, add 2 µL of diluted DNA directly to the OLA plate without drying it down (wet gDNA method).IMPORTANT! Do not dispense DNA into wells that are designated NTC and allelic ladder.
C
T
G
A
C GT A
Step Step Type Temperature ( ° C) Time
1 Hold 4 1 min
2 Hold 99 5 min
3 Hold 4 ∞
Page 6
2 Phosphorylate and ligate the probes (OLA).
a. Prepare an OLA reaction mix by scaling the volumes listed below to the desired number of OLA reactions.
b. Retrieve and label the appropriate number of reaction plates.
c. Prepare the OLA reaction by adding the following components to each well of a 384- or 96-well plate.IMPORTANT! Do not add reaction mix to the allelic ladder wells.
GeneAmp® PCR System 9700 Thermal Cycler
F1 F2 F3 F4
1 2 3
4 5 6
7 8 9
ENTER
STOP
0 CE
F5
GeneAmp®
PCR System 9700
POWER
CG
C
LSO LinkerASOL2
ASOA2 LSO
OLA Master Mix SNPlexSystem, Universal Linkers,48-plex SNPlex System
ASOA2
ASOA1
LSO
LSO linkerASOL1
ASOL2
C
G
Component Dried-down gDNA (µL) Wet gDNA (µL)
Nuclease-free water 2.30 0.30
OLA Master Mix (2✕) SNPlex System
2.50 2.50
Universal Linkers, 48-plex 0.05 0.05
SNPlex Ligation Probes 0.10 0.10
dATP (100✕) SNPlex System 0.05 0.05
Total 5.00 3.00
C = Control DNA, NTC = No Template Control, L = Allelic Ladder
96-capillary array, 96-well plate 96-capillary array, 384-well plate
48-capillary array, 96-well plate 48-capillary array, 384-well plate
16-capillary array, 96-well plate 16-capillary array, 384-well plate
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
NTC
L
C
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
CNTC
L
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
NTC
L
C
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
CNTC
L
If the plate contains dried-down gDNA (Dried-down gDNA method)...
If gDNA has not been added to the plate (Wet gDNA method)...
5 µL OLA reaction mix 3 µL OLA reaction mix
2 µL gDNA (see step 1 on page 6)
Page 7
d. Use a recommended cover and compression pad to cover the plate, then run the thermal cycler (The conditions below are for an Applied Biosystems Dual 384-Well GeneAmp® 9700 PCR System.):
Note: The OLA Master Mix contains enzymes to promote phosphorylation of probes and linkers, UNG to degrade contaminating amplicons, and ligase. All steps are carried out sequentially during the thermocycling protocol. Note: The OLA reaction is optimized for the Applied Biosystems GeneAmp® 9700 PCR System. If a different thermal cycler is used, refer to the conditions specified in the SNPlex™ Genotyping System 48-plex User Guide.IMPORTANT! Applied Biosystems has found that some plate covers negatively affect the performance of the SNPlex Assay. It is critical to use a recommended plate cover that will not affect the chemistry of the ligation reaction. See the SNPlex™ Genotyping System 48-plex User Guide for recommended plate covers.
e. Proceed directly to exonuclease digestion. Alternatively, you can store OLA reactions at −20 ° C for up to 21 days.
3 Purify ligation product by exonuclease digestion.
a. Directly before use, prepare a 2✕ Exonuclease master mix on ice by scaling the volumes listed below to the desired number of OLA reactions. Prepare extra volume to account for pipetting losses.
b. Pipette 5.0 µL of 2✕ Exonuclease master mix into each well of the OLA reaction. Cover the plate, then vortex and spin briefly.
c. Transfer the reaction plate to the thermal cycler, and start the program.
d. Proceed to dilution of the Exonuclease reactions and PCR amplification. Note: The reactions can either be left at 4 ° C overnight in the thermal cycler, or stored at −20 ° C for up to 21 days.
wi
Step Step Type Temperature ( ° C) Time
1 HOLD 48 30 min
2 HOLD 90 20 min
3 25 Cycles 94 15 sec
60 30 sec
513% rampa
a Use a 2% ramp with standard or maximum setting for 96-well single plate modules on the Applied Biosystems 9700 GeneAmp® 9700 PCR System. Use a 3% ramp for the dual 96-well module.
30 sec
4 HOLD 99 10 min
5 HOLD 4 ∞
Component Volume per Reaction (µL)
Nuclease-free water 4.2
Exonuclease Buffer, SNPlex System (10✕)
0.5
Lambda Exonuclease, SNPlex System 0.2
Exonuclease I, SNPlex System 0.1
Total 5.0
Step Step Type Temperature ( ° C) Time
1 Hold 37 90 min
2 Hold 80 10 min
3 Hold 4 ∞
Page 8
4 PCR amplify the ligated and exonuclease digested products.
a. Dilute the 10 µl Exonuclease-treated ligation reactions with 15 µl of nuclease-free water to each well and mix to combine. Note: If less than the recommended amount of DNA is being used, consider adding less water prior to PCR.
b. Prepare a PCR master mix by scaling the volumes listed below to the desired number of PCR reactions. Prepare extra volume to account for pipetting losses.
c. Dispense the following into each well: 8.0 µL PCR master mix + 2.0 µL diluted OLA reaction product.
d. Seal the plate with a recommended cover. Vortex the plate briefly, then spin down.Note: Applied Biosystems recommends that you perform all subsequent steps in a different laboratory to avoid amplicon contamination.
e. Transfer the reaction plate to the thermal cycler and start the program.
f. Proceed to bind the amplicons to a streptavidin-coated plate immediately. Note: You can store the reactions at −20 ° C for up to 35 days.
5 Bind PCR products to hybridization plate, then isolate biotinylated strand.
IMPORTANT! For best results, steps 5 through 7 must be executed continuously.
a. Wash the wells of the SNPlex Hybridization Plate once with 100 µL of Wash Buffer diluted 1:10 with deionized water.
b. Add 0.009 µL positive hybridization control to 17.491 µL Binding Buffer.
c. Add 17.5 µL of the Binding Buffer containing positive hybridization control to the SNPlex Hybridization Plate.
d. Transfer 3.0 µL of PCR product to the SNPlex Hybridization Plate, then mix and cover the plate. Incubate at room temperature for 15 to 60 min on a rotary shaker.Note: The notches on plates from different manufacturers do not always line up. Make sure you orient the plates properly—with well A-1 on the upper left corner—when transferring samples between plates.
e. Briefly spin the plate and then add 50 µL of 0.1 N NaOH. Incubate for 5 to 30 min at room temperature on a rotary shaker.
f. Briefly spin the plate and remove the supernatant, then wash three times with 100 µL Wash Buffer diluted 1:10 with deionized water.
Component Volume per Reaction (µL)
Nuclease-free water 2.5
Amplification Master Mix (2X) SNPlex System
5.0
Amplification Primers (20X) SNPlex System
0.5
Total Volume 8.0
Step Step Type Temperature ( ° C) Time
1 Hold 95 10 min
2 30 cycles 95 15 sec
63 1 min
3 Hold 4 ∞
GR2371SNPlex
PCR Hybridization Electrophoresis
Page 9
6 Hybridize the ZipChute™ probes to the ZipCode™ sequences.
a. Equilibrate the oven to 37 ° C, then prepare a hybridization master mix by scaling the volumes listed below to the desired number of samples. Prepare extra volume to account for pipetting losses.
b. Add 25 µL of the hybridization master mix to each well of the SNPlex Hybridization Plate, then cover. Note: Do not use the MicroAmp™ 96-well full plate cover for this step. Use one of the other recommended plate covers (part number N8010550) from Table 1-2 of the SNPlex™ Genotyping System 48-plex User Guide.
c. Incubate the plate for 60 to 75 min at 37 ° C on a rotary shaker. Note: During incubation, do not expose the plate to bright light.
7 Prepare size standards, elute the ZipChute™ probes, and dispense the allelic ladder.
a. Prepare a Sample Loading Mix by scaling the volumes listed below to the desired number of samples. Prepare fresh sample loading mix daily.
b. Briefly spin the plate and remove the supernatant, then wash three times with 100 µL Wash Buffer diluted 1:10.IMPORTANT! The ZipChute probes may be stripped off the plate under the following conditions:– Rapid aspiration of the ZipChute Mix or Wash Buffer supernatant when using a plate washer– Contact between the plate washer tips and the well surfacesApplied Biosystems recommends that you set the aspiration tip depth so that 15 to 20 µL of Wash Buffer remains in each well after each aspiration, preventing the tips from touching the bottom of the wells. For more information about configuring plate washers, refer to the SNPlex™ Genotyping System 48-plex General Automation Getting Started Guide, or the SNPlex™ Genotyping System 48-plex Automation Guide Automating PCR Using the Tomtec Quadra 3 Getting Started Guide.IMPORTANT! After the final wash, spin the plate upside down at 1000 rpm for 60 sec on a stack of paper towels.
c. Add 17.5 µL of Sample Loading Mix into each well and mix.
d. Cover the plate and incubate at 37 ° C for 30 min on a rotary shaker.
ZipChute Dilution Buffer, SNPlex SystemDenaturant, SNPlex System ZipChuteTM Mix, SNPlex System
Remove supernatant
B
G
Component Volume per Reaction (µL)
ZipChute Mix, SNPlex System 0.05
Denaturant, SNPlex System 11.25
ZipChute Dilution Buffer, SNPlex System 13.70
Total 25.00
Size Standard, 48-plex SNPlex SystemSample Loading Reagent, SNPlex System
B
Applied Biosystems 3730/3730xl/ 3130xl DNA Analyzer
Component Volume per Reaction (µL)
Size Standard, 48-plex SNPlex System 0.59
Sample Loading Reagent, SNPlex System 16.91
Total 17.5
Page 10
8 Prepare samples for electrophoresis.
To dispense the allelic ladder and transfer reagent from the hybridization plate:
a. Remove the hybridization plates from the oven.
b. Briefly spin the plates to collect the fluid at the bottom of the wells.
c. Label a new reaction plate. The reaction plate must be appropriate for use with Applied Biosystems 3730/3730xl and 3130xl DNA Analyzers.
d. If using 384-well plates, transfer 7.5 µL from each well into the wells of the new plate.
If using 96-well plates, transfer 10 µL from each well into the wells of the new plate.
e. Load the Allelic Ladder wells as shown in dark blue in the figure below:Note: The Allelic Ladder is part of the SNPlex System Standards Kit.
Note: Make sure there are no air bubbles trapped at the bottom of the wells. If there are bubbles, briefly spin the plate.Note: For information about proper sample plate layout, refer to “Designing the Sample Plate Layout” in the SNPlex™ Genotyping System 48-plex User Guide.IMPORTANT! If you are not going to immediately use the plates for analysis, seal the plates and store at −20 ° C.Note: Consider the plate seal options for use with the 3730 and 3730xl instruments. While both septa and heat seal film are available, the septa do not provide an air-tight seal. Some gradual signal loss occurs over time when using the septa. If the SNPlex™ plates will remain on the instrument in excess of 12 hours, Applied Biosystems recommends using the pierceable heat seal option. (Heat Seal film, 3730/3730xl instrument only; septa only can be used with the 3130xl instrument.) Be aware that after the heat seal is pierced by the instrument for sample injection, the seal is no longer intact.
96-capillary array, 96-well plate 96-capillary arrays, 384-well plate
48-capillary array, 96-well plate 48-capillary array, 384-well plate
16-capillary array, 96-well plate 16-capillary array, 384-well plate
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12 Each well contains 10 µL from hybridization plate
1.25 µL Allelic Ladder
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Each well contains 7.5 µL from hybridization plate
1 µL Allelic Ladder
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12 Each well contains 10 µL from hybridization plate
1.25 µL Allelic Ladder
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Each well contains 7.5 µL from hybridization plate
1 µLAllelic Ladder
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12 Each well contains 10 µL from hybridization plate
1.25 µL Allelic Ladder
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Each well contains 7.5 µL from hybridization plate
1 µL Allelic Ladder
Page 11
9 Create a results group for SNPlex System experiments.
a. In the Data Collection software, open the Results Group Editor, then select the Naming tab.
b. Complete the information on the tab as shown below:
Note: This figure shows the Results Group Editor for instruments running 16- or 48-capillary arrays. For instruments running 96-capillary arrays, the Run Sequence Number field is not necessary.IMPORTANT! For GeneMapper software to correctly process SNPlex System data, run folder naming conventions and sample plate layout (as shown in “Sample Plate Layout Examples” on page 2) must be compatible.
10 Create the plate record. Note: The following procedure explains how to create plate records using the fully automated workflow recommended for high-throughput environments. Refer to the SNPlex™ Genotyping System 48-plex User Guide for information about setting up plate records manually.
a. In the Data Collection software, open the Plate Manager.
b. Click Import, then navigate to the text file that you want to import.
c. Select the file that you want to import, then click Open.
The Data Collection software imports the contents of the file into a new plate record and displays a confirmation message if the import is successful.
IMPORTANT! For GeneMapper software to recognize the SNP set information, you must have imported the assay information file into GeneMapper software (Step 2, “Load the SNPlex™ System panels and bins.” on page 13).
The following table summarizes SNPlex System plate record information.
Parameter Value
Sample Name Name entered in the Data Collection software.
Sample Type Sample type entered in the Data Collection software.
Size Standard SNPlex_48plex_size_standard_v1
Panel SNPlex_48plex_Panel_3730 or SNPlex_48plex_Panel_3130
Analysis Method SNPlex_Rules_3730 or SNPlex_Model_3730 or SNPlex_Rules_3130
SNP Set Imported from assay information file. (See “Import the AIF.” on page 13.)
Results Group See “Create a results group for SNPlex System experiments.” on page 12.
Instrument Protocol Instrument protocol for SNPlex system experiments associates HTSNP36_POP7_V2.xml and Dye Set S.
Note: For Data Collection v3.0, this is HTSNP36_POP7_V3 or HTSNP50_POP7.
Page 12
Analyzing SNPlex System Data in GeneMapper Software v4.0
11 Perform electrophoresis. a. Load the plate into the instrument, ensuring that the plate assembly fits flat in the stacker/autosampler.
b. Run the plate.
Next, analyze your data (see “Analyzing SNPlex System Data in GeneMapper Software v4.0”).
1 Install GeneMapper Software v4.0.
Note: This is a one-time setup step.
Install GeneMapper software v4.0 according to instructions provided in the GeneMapper® Software v4.0 Installation Guide.
2 Load the SNPlex™ System panels and bins.
Note: This is a one-time setup step. All the files are installed with the software but they must be imported before analyzing SNPlex System data for the first time.
a. Start the GeneMapper software v4.0.
b. Click (Panel Manager), then import the SNPlex System:– Panels: SNPlex_48plex_3730_Panels.txt or SNPlex_48plex_3130_Panels.txt– Bins: SNPlex_48plex_3730_Bins.txt or SNPlex_48plex_3130_Bins.txt
c. Close the Panel Manager.
3 Import the AIF. a. In the SNP Sets tab of the GeneMapper Manager, click Import.
b. Insert the SNPlex Genotyping System Ligation Probes CD, then select the AIF (SNPlex_nnnnnnn_nnnnnnn.xml).IMPORTANT! For GeneMapper software to read SNP sets from the plate record, you must import the AIF into GeneMapper software before you import the sample files.IMPORTANT! If you are using the SNPlex_Model_3730 or any analysis method that uses the Model clustering algorithm, you must import the AIF into GeneMapper software.
4 Import SNPlex System data into GeneMapper software.
a. Before GeneMapper software can analyze SNPlex System data, you must complete the following fields: – Sample Name– Sample Type– SNP Set– Analysis Method– Panel– Size Standard.Note: Depending on the way you set up your plate records, some or all these fields may already be complete.
b. Select File > Add samples to project to add the sample files.
c. Select the data that you want to analyze, then click Add to list.
d. After adding all relevant files, click Add to add the files to the project.
5 Analyze the data and review the results.
a. Before proceeding with analysis, check to see that:– Samples have the correct sample type designation– Analysis Method is set to SNPlex_Model_3730 or SNPlex_Rules_3730 or
SNPlex_Rules_3130 for all samples– Panel is set to SNPlex_48plex_3730_Panels or SNPlex_48plex_3130_Panels for all samples– Size Standard is set to SNPlex_48plex_v1 for all samples– SNP set is set to the appropriate SNP set for each sample
b. Click (Analysis > Analyze Samples).
Data Collection Software
GeneMapperR Software v4.0
Applied Biosystems 3730/3730xl/ 3130xl DNA Analyzer
Page 13
SNPlex Genotyping System, 48-plex Required Equipment and Consumables
Table 1 SNPlex Genotyping System, 48-plex Required Equipment and Consumables
Item Vendor Part Number
Applied Biosystems 3730/3730xl DNA Analyzer See your Applied Biosystems representative for information.
Consumables POP-7™ polymer, 28-mL bottle Applied Biosystems
4363929
POP-7 polymer, 28-mL bottle, box of 10
4363935
DS-40 Spectral Calibration Standard Kit (Dye Set S)a
4349365
10✕ Running Buffer with EDTA 4335613
36-cm 48-capillary array 4331247
50-cm 48-capillary array 4331250
36-cm 96-capillary array 4331244
50-cm 96-capillary array 4331246
96-Well Plate Septa 4315933
384-Well Plate Septa 4315934
Plate Accessories
96-Well Plate Base (septa sealed) Applied Biosystems
4334873
96-Well Plate Retainer (septa sealed) 4334869
384-Well Plate Base (septa sealed) 4334874
384-Well Plate Retainer (septa sealed) 4334868
96-Well Plate Base (heat sealed) 4334875
384-Well Plate Base (heat sealed) 4334877
96- and 384-Well Plate Retainer (heat sealed)
4334865
Page 14
Applied Biosystems 3130xl DNA Analyzer See your Applied Biosystems representative for information.
Consumables POP-7 polymer, 7-mL bottle Applied Biosystems
4352759
POP-7 polymer, 3.5-mL bottle 4363785
DS-40 Spectral Calibration Standard Kit (Dye Set S)a
4349365
10✕ Running Buffer with EDTA 4335613
36-cm 16-capillary array 4315931
50-cm 16-capillary array 4315930
96-Well Plate Septa 4315933
384-Well Plate Septa 4315934
Plate Accessories
96-Well Plate Base Applied Biosystems
4317237
96-Well Plate Retainer 4317241
384-Well Plate Base 4317236
384-Well Plate Retainer 4317240
GeneAmp® PCR System 9700 Dual 384-Well Sample Block Module orGeneAmp® PCR System 9700 Dual 96-Well Sample Block Module
See your Applied Biosystems representative for information.
Reaction Plates
MicroAmp™ Optical 96-Well Reaction Plate
Applied Biosystems
N8010560
ABI PRISM® 384-Well Clear Optical Reaction Plate, with Barcode, 50 plates
4309849
ABI PRISM® 384-Well Optical Reaction Plate with Barcode, 500 plates
4326270
96-Well Sample Plates w/barcode 4306737
Reaction Plate Coversb
MicroAmp™ 96-Well Full Plate Coverc
(Do not use for post-PCR steps)Applied Biosystems
N8010550
ABI PRISM® Optical Cover Compression Pad (requires adhesive and heat seals).
IMPORTANT! Do not use compression pads with MicroAmp™ 96-Well Full Plate Covers.
4312639
Adhesive Sealsb
384-Well Microplate Aluminum Sealing Tape
Corning 6569
Adhesive PCR Foil Seal ABGene AB-0626
Silverseal Greiner 676 090
Table 1 SNPlex Genotyping System, 48-plex Required Equipment and Consumables (continued)
Item Vendor Part Number
Page 15
Heat Seals and Sealersb
Easy-Peel 610 Meter Roll ABGene AB-3739
Easy-Peel Individual Sheets AB-0745
Thermo-Sealer AB-0384
Plate Sealer, ALPS 300 AB-0950
Uniseal AL Whatman 7704-0002
GeneMapper® Software v4.0d See your Applied Biosystems representative for information.
Data Collection Software v2.0 or higher See your Applied Biosystems representative for information.
a Provided in the SNPlex System Starter Kit.b IMPORTANT! Applied Biosystems has found that certain plate covers negatively affect the performance of the
SNPlex System assay. If you use covers other than the recommended plate covers, test them using the SNPlex™ System Control Set (see Appendix A).
c Because they do not seal without pressure in a thermal cycler, do not use MicroAmp™ 96-Well Full Plate Covers in hybridization steps.
d Modules for GeneMapper and Data Collection Software are available at http://www.appliedbiosystems.com/support/software.
Table 1 SNPlex Genotyping System, 48-plex Required Equipment and Consumables (continued)
Item Vendor Part Number
Page 16
SNPlex System Kits and Reagents
Table 2 SNPlex System Kits and Reagents
Reagent Name Part Number
SNPlex™ Genotyping System Core Reagents Kita, 48-plex (5000 reactions)
4362266
SNPlex™ System Core Kit (1500 reactions) 4375768
SNPlex™ System Assay Control Kita
Control DNA SNPlex™ Systemb
4349363
SNPlex™ System Oligonucleotide Ligation Kita
• Universal Linkers, 48-plex SNPlex™ System • Oligonucleotide Ligation Master Mix SNPlex™ System• dATP (100✕) SNPlex™ System
4362268
SNPlex™ System Purification Kita
• Lambda Exonuclease SNPlex™ System • Exonuclease Buffer (10✕) SNPlex™ System • Exonuclease I SNPlex™ System
4349357
SNPlex™ System Amplification Kita
• Amplification Primers (20✕) SNPlex™ System • Amplification Master Mix (2✕) SNPlex™ System
4349358
Hybridization Binding Buffer SNPlex™ System 4349304
Hybridization Wash Buffer (10✕) SNPlex™ System 4349301
ZipChute™ Dilution Buffer SNPlex™ System 4349306
SNPlex™ System ZipChute™ Kit, 48-plex
• Denaturant SNPlex™ System• ZipChute™ Mix, 48-plex SNPlex™ System • Positive Hybridization Controls SNPlex™ System
4349361
SNPlex™ System Standards Kit, 48-plex
• Sample Loading Reagent SNPlex™ System • Size Standard, 48-plex SNPlex™ System • Allelic Ladder, 48-plex SNPlex™ System
4349351
SNPlex™ System Starter Kit, 48-plex 4362267
SNPlex™ Genotyping Dried gDNA Plate Control Pool System CD 4366107
SNPlex™ Genotyping System 48-plex User Guide 4340856
SNPlex™ Genotyping System 48-plex Quick Reference Card 4340855
SNPlex™ Genotyping System 48-plex General Automation Getting Started Guide
4363143
SNPlex™ Genotyping System 48-plex Automation Guide Automating PCR Using the Tomtec Quadra 3 Getting Started Guide
4358100
SNPlex™ System Array Conditioning Kit 4352018
SNPlex™ System Control Pool, 48-plex 4362635
Page 17
SNPlex™ System Dried gDNA Plates 4362637
DS-40 Spectral Calibration Standard Kit (Dye Set S) 4349365
SNPlex™ System Control Pool Kit
• Control Pool, 48-plex SNPlex™ System• SNPlex™ Genotyping Dried gDNA Plate Control Pool System CD
4362639
SNPlex™ System Dried gDNA Plates Kit
• Dried gDNA Plate SNPlex™ System• SNPlex™ Genotyping Dried gDNA Plate Control Pool System CD
4366135
SNPlex™ System Hybridization Plates, 384-well (5 plates) 4349369
SNPlex™ System Hybridization Plates, 96-well (5 plates) 4357279
SNPlex™ System Hybridization Plates, 96-well (10 plates) 4362933
SNPlex™ System Ligation Probes 4346978
a
a Each SNPlex System kit provides sufficient reagent to perform 5,000 reactions on the 3730. A smaller System Core Kit is available for performing 1,500 reactions. If you do not expect to consume into aliquots all the reagents in a kit in a single use, Applied Biosystems recommends that you divide the remaining reagents into aliquots to minimize repeated freeze-thaw cycles. Components of the core reagent and starter kits can be ordered individually.
b Sufficient for 5,000 reactions (Applied Biosystems 3730xl, 3130xl DNA Analyzer) or 2,500 reactions (Applied Biosystems 3730 DNA Analyzer).
Table 2 SNPlex System Kits and Reagents (continued)
Reagent Name (continued) Part Number
Page 18
NOTICE TO PURCHASER: PLEASE REFER TO THE SNPlex™ Genotyping System 48-plex User Guide (PN 4360856) FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION.
Applera, Applied Biosystems, AB (Design), GeneAmp, GeneMapper, and ZipChute are registered trademarks and MicroAmp, SNPlex, and ZipCode are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries.
9/2007
© Copyright 2007, Applied Biosystems. All rights reserved
www.appliedbiosystems.com Part Number 4360855D