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The figures below illustrate the arrangement of the standard and unknown populations
on the RNase P plate. The RNase P plate contains five replicate groups of standards
(1250, 2500, 5000, 10,000, and 20,000 copies), two unknown populations (5000 and
10,000 copies), and four no template control (NTC) wells.
After the run, the SDS software:
The instrument passes the verification if the analyzed data demonstrates that the
instrument distinguishes between 5,000 and 10,000 genome equivalents with a 99.7%
confidence level.
IMPORTANT!
TaqMan RNase P Instrument Verification Plate can be omitted from the analysis to meet
specifications.
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Installation and Maintenance Guide
5000-copy Unknown
Population 1
NTC
STD 1250
STD 2500
STD 5000
STD 10000
STD 20000
GR2341
Gemini
10000-copy Unknown
96-well plate one sample blue
Population 2
Plate for 7300/7500 system
• Generates a standard curve from the averaged threshold cycle (C
replicate groups of standards.
• Calculates the concentration of the two unknown populations using the standard
curve.
• Calculates the following using the mean quantity and standard deviation for the
5,000- and 10,000-copy unknown populations to assess the instrument
performance:
[
(
) 3 σ
(
CopyUnk
–
2
where:
• CopyUnk
= Average copy number of unknown #1 (5,000-copy population)
1
• σ
= Standard deviation of unknown #1 (5,000-copy population)
CopyUnk1
• CopyUnk
= Average copy number of unknown #2 (10,000-copy population)
2
• σ
= Standard deviation of unknown #2 (10,000-copy population)
CopyUnk2
Up to six outlier wells from each unknown replicate group in a 96-well
Chapter 7 Verifying the Instrument Performance
1
2
3
4
A
B
C
D
NTC
E
STD 5000
F
GR2478b
7500
G
96-well plate one sample blue
H
Plate for 7500 Fast system
)
]
[
(
)
>
CopyUnk
+
CopyUnk2
1
Overview
5
6
7
8
9 10 11 12
RNase P
Population 1
STD 1250
STD 2500
STD 10000
STD 20000
RNase P
Population 2
) values of the
T
3 σ
(
)
]
CopyUnk1
7
89
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