BGI Real-Time Fluorescent RT-PCR Kit for detecting 2019-nCoV

Materials:

 P200
 P100
 P20
 P10
 Filtered pipette tips
 8-strip PCR tubes, low profile with optical strip cap
 Cooling rack for 1.5 ml tubes and PCR tubes
 Centrifuge for 8-strip PCR tubes

A. PCR Mix:
*Perform this on the chilled PCR rack

Component 1x (30µL) x
2019-nCoV Reaction Mix 18.5 µL
2019-nCoV Enzyme Mix 1.5 µL
sample RNA/positive 10 µL -
control/NTC
Total 30 µL ____ µL

1. Prepare the PCR mix in well 1 of the 8-tube PCR strip that is good for _____ number of
samples including the positive control and NTC. DO NOT write on the sides of the tubes.
2. Distribute 20 µL of master mix into empty tubes.
3. Add 10 µL samples/positive control/NTC to the tubes following the layout as shown below
and pipet mix:

1 2 3 4 5 6 7 8
U U U NTC NTC NTC NTC NTC

4. Seal the tubes and centrifuge for 10 seconds. DO NOT write the tops/lids of the tubes.
You may use the figure above to use as legends for the tubes.
B. Real-Time PCR:

Channels: FAM (for RNA of 2019-nCoV) and VIC/HEX (for internal reference)
Reference dye: None
Sample volume: 30 µL

PCR Profile
Step Cycle Temperature Duration Fluorescence Measurement?
1 1 cycle 50°C 20 minutes No
2 1 cycle 95°C 10 minutes No
95°C 15 seconds No
3 40 cycles
60°C 30 seconds Yes