Download Clostridium difficile PCR Detection Kit - Protocol

C. PCR Assay Programming
1. Program the thermocylcer according to the program shown in Table 4 below.
2. Run PCR.
Table 4. C. difficile Assay Program
One Step PCR Cycle
Cycle 1
Cycle 2 (35x)
Step
Cycle 4
o
Duration
Step 1
95 C
5 min
Step 1
94oC
15 sec
Step 2
Step 3
Cycle 3
Temperature
o
30 sec
o
45 sec
o
5 min
60 C
72 C
Step 1
72 C
Step 1
o
4C
∞
D. Clostridium difficile PCR Assay Results Interpretation
1. For the analysis of the PCR data, the entire 15-20 µL PCR Reaction should be loaded on a
1X TAE 1.7% Agarose DNA gel along with 10 µL of Norgen’s DNA Marker (provided). Prepare
enough agarose gel for running one set of PCR of C. difficile detection and one set of PCR for
controls detection.
2. The PCR products should be resolved on the 1X TAE 1.7% Agarose gel at 150V for 30
minutes (Gel running time will be vary depending on an electrophoresis apparatus).
3. Sample results are provided below:
M
2000
1500
1000
750
500
300
150
C. difficile
NC
C. difficile Target
Figure 1: A representative 1X TAE 1.7% agarose gel showing the amplification of C. difficile (C. difficile
Target) using the 2X C. difficile PCR Master Mix. The size of the C. difficile target amplicon corresponds to
325 bp as represented by the provided DNA Marker (M). NC = Negative Control.