COVID-19 RBD amplification protocol
1. Prepare a master mix using the following reagents in a sterile Biosafety cabinet.
Reagent
2X Platinum SuperFi RT-PCR Master
Mix*
SuperScript IV RT Mix*
Fw primer – MCJ2395 (50 µM)
Rv primer – MCJ2452 (50 µM)
Nuclease free water
RNA template from wastewater
extraction
*Invitrogen, Catalog #: 12594100
B.
C.
D.
E.
F.
G.
H.
Volume per reaction
(µl)
12.5
0.25
0.25
0.25
6.75
5
2. Preform primary
amplification in a
thermocycler using the
steps listed below.
A. RT enzyme activation:
25°C for 2 minutes
Reverse transcription: 50°C for 20 minutes
RT inactivation/Initial denaturation: 95°C for 2 minutes
Denaturation: 95°C for 15 seconds
Annealing: 55°C for 30 seconds
Extension: 72°C for 1 minute
Repeat steps D – F for 25 cycles
Hold at 4°C
3. Prepare master mix for a nested PCR using the following reagents.
Reagent
Q5 High-Fidelity DNA Polymerase*
Q5 Reaction Buffer*
Deoxynucleotide Solution Mix# (10 mM for each
dNTP)
Fw primer – MCJ2469 (50 µM)
Rv primer – MCJ2370 (50 µM)
Nuclease free water
DNA template from primary reaction
*New England Biolabs, Catalog #: M0491L
# New England Biolabs, Catalog #: N0447S
Volume per reaction
(µl)
0.125
4
0.25
0.25
0.25
15.125
5
4. Preform nested amplification in a thermocycler using the steps listed below.
A. Initial denaturation: 95°C for 2 Minutes
B. Denaturation: 95°C for 15 seconds
C. Annealing: 55°C for 30 seconds
D. Extension: 72°C for 1 minute
E. Repeat steps B – D for 20 cycles
F. Hold at 4°C
5. Confirm amplification through gel electrophoresis. Expected amplicon size is ~600bp
MCJ2395 CTGCTTTACTAATGTCTATGCAGATTC
MCJ2452 NCCTGATAAAGAACAGCAACCT
MCJ2469 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTRATGAAGTCAGMCAAATYGC
MCJ2370 gtgactggagttcagacgtgtgctcttccgatctATGTCAAGAATCTCAAGTGTCTG