Biomedical Engineering Reference
In-Depth Information
final reaction volume of 25
µ
L. Add 50 aliquots of 23
µ
L into a 96-well PCR
microplate.
2.
Add 5 pmol each of forward (PCR-F) and reverse (PCR-R) series of primers for
each of the 50 reactions amplifying the overlapping amplicons that cover the whole
SARS-CoV genome (
see
Note 7
). The primer sequences are shown in
Table 4
.
3.
In an area separate from the hood dedicated for PCR, add 1
µ
L of diluted reverse-
transcribed products.
4.
Commence with PCR in a thermocycler with initial denaturation at 95°C for
1 min and 35 cycles of 95°C for 0.5 min, 55°C for 0.5 min, 68°C for 1.5 min, and
a final extension at 68°C for 10 min.
3.5. Secondary PCR Amplification
1.
Inside a hood dedicated for setting up PCR, assemble the PCR master mix for the
50 reactions according to
Table 3
in a final reaction volume of 25
µ
L. Add 50
aliquots of 23
µ
L into a new 96-well PCR microplate.
2.
Add 5 pmol each of forward (PCR-F) and reverse (BSEQ-R) series of primers for
each of the 50 semi-nested PCR reactions. The primer sequences are shown in
Table 4
.
3.
In an area separate from the hood dedicated for PCR, add 1
µ
L of the correspond-
ing primary PCR product.
4.
Commence PCR in a thermocycler with initial denaturation at 95°C for 1 min
and 35 cycles of 95°C for 0.5 min, 55°C for 0.5 min, 68°C for 1.5 min, and a final
extension at 68°C for 10 min.
5.
Electrophorese 5
L of the secondary PCR product in a 2% agarose gel to verify
the success of the PCR amplification. Estimate the amount of PCR product by
comparison to DNA marker. Only products with single band should be used for
sequencing.
µ
3.6. Cycle Sequencing
Perform sequencing reaction based on the dideoxy dye terminator method,
according to manufacturers' instructions:
1.
Separate from the hood dedicated for PCR, assemble the cycle sequencing reac-
tion with ASEQ-F, BSEQ-F, ASEQ-R, and BSEQ-R series of oligonucleotides
as sequencing primers for each of the amplicon, and with 2-5 ng of secondary
PCR product as sequencing template (
see
Note 8
).
2.
Commence with cycle sequencing reaction in a thermocycler.
3.
Purify the extension products with either spin column purification or ethanol pre-
cipitation. Mix or resuspend the DNA in formamide solution according to the
manufacturer's instructions.
4.
Denature the purified extension products at 95°C for 5 min, snap-cool on ice, and
load onto the automated capillary DNA sequencer for injection.
