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  3. TaqMan® Pathogen

Thermo Fisher Scientific TaqMan® Pathogen Owner's manual

  • Hello! I've reviewed the user manual for the Applied Biosystems TaqMan Pathogen Detection Kit. This document provides instructions for setting up real-time PCR reactions, including preparing the master mix, adding controls, and setting up thermal cycling conditions. I can help you with questions about the procedure, analysis, and interpretation of results.
  • What is the recommended volume of pathogen PCR cocktail to dispense into each well?
    How much sample DNA is added to each designated well?
    How much Positive Control is added to each control well?
BIOHAZARD. Biological samples such as tissues and blood have the potential to transmit
infectious diseases. Read and follow the guidelines published in Biosafety in Microbiological and Biomedical
Laboratories (http://bmbl.od.nih.gov) and in the Occupational Safety and Health Standards, Bloodborne pathogens
(http://www.access.gpo.gov/nara/cfr/waisidx_01/29cfr1910a_01.html). Wear appropriate protective eyewear,
clothing, and gloves.
STEP
5 µL Negative Control
ACTION
TaqMan
®
Pathogen
Detection Kit
Reaction Setup
A. Make the pathogen PCR Cocktail ( ) as shown in this table.
B. Dispense 45 µL of the pathogen PCR Cocktail into all of the
designated wells.
• Add
into each of the two negative control wells.
• Cap the wells.
• Add
into each of the designated UNKN ( ) wells.
Note: This example shows 54 sample reactions
with four control reactions.
• Cap the wells.
Number of
Reactions
Total
Volume
Pathogen
PCR
Mix (µL)
AmpliTaq Gold®
DNA Polymerase
(µL)
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
484
748
968
1188
1452
1672
1936
2156
2376
2640
2860
3124
3344
3564
3828
4048
4312
495
765
990
1215
1485
1710
1980
2205
2430
2700
2925
3195
3420
3645
3915
4140
4410
11
17
22
27
33
38
44
49
54
60
65
71
76
81
87
92
98
5 µL sample DNA
A
B
C
D
E
F
G
H
12 3 4 5 6 7 8 9 10 11 12
Pos
Control
Neg
Control
A
B
C
D
E
F
G
H
12 3 4 5 6 7 8 9 10 11 12
Pos
Control
Neg
Control
A
B
C
D
E
F
G
H
12 3 4 5 6 7 8 9 10 11 12
Pos
Control
Neg
Control
A
B
C
D
E
F
G
H
12 3 4 5 6 7 8 9 10 11 12
Pos
Control
Neg
Control
5 µL Positive Control
• Add
into each of the two positive control wells.
• Cap the wells.
P/N 4330677 Rev. A
This example shows the plate
setup for qualitative (pathogen
presence or absence) analysis
mode.
For quantitative analysis mode,
refer to the User Guide,
Chapters 3, 4, and 6.
TaqMan
®
Pathogen
Detection Kit
Procedure Flowchart
See the reverse side of this card for details
See Product Insert for details
See PrepMan Ultra Protocol
(P/N 4318925) for details
Open the real-time stationery file
Designate sample types: UNKN for FAM layer, IPC+ for VIC layer
Click Plate Setup and designate replicate name
Check thermal cycling conditions
Save the file (do not use Save as…)
P/N 4330677 Rev. A
Instrument Setup
Reaction Setup
Open Experiment
Report Window
PCR Thermal Cycling
PrepMan™ Ultra Sample
Preparation Reagent
DNA Purification
Pre-Enrichment
9 min.
94 ˚C
20 sec.
95 ˚C
Stage 1 Stage 2
1 min.
60 ˚C
Repeat 40
30 sec.
72 ˚C
Click Show Analysis
Click Run
Save the file after the run is finished
From the Window menu, select Experiment Report
From the Analysis menu, select Analyze
Optional
Initial Analysis
0.5–1 hr
15–30 min
10–20 min
2.5 hrs
2 min
Variable
16–24 hrs
2 min
Optional
Sample
Examine the Experiment Report as follows:
For each sample, note the CT FAM result under the CT column heading
For each sample, also note the presence/absence of the words “No Amp”
under the Quantity column heading
Score results according to the following table:
Final Analysis
© Copyright 2001, Applied Biosystems. All rights reserved.
“CT” Column “Quantity” Column Assay Result
CT < 40
CT= 40
CT= 40
Either
“No Amp” appears
or
“No Amp” does not appear
“No Amp” does not appear
“No Amp” appears
Positive
Negative
No Amplification
(re-test sample)
PrepManTM
Ultra
/