Biomedical Engineering Reference
In-Depth Information
Table 2
Composition of Reaction Mix for Reverse-Transcription
of Severe Acute Respiratory Syndrome-Coronavirus RNA
Component
Volume for one reaction (
µ
L)
Final concentration
5X first strand buffer
4
1X
dNTP mix (10 m
M
each dATP, dGTP,
dCTP, and dTTP
at neutral pH)
1
0.5 m
M
each
0.1
M
dithiothreitol 1
5 m
M
RNasin RNase inhibitor
1
2 U/
µ
L
L)
SuperScript III reverse
(40 U/
µ
2
20 U/
µ
L
transcriptase (200 U/
µ
L)
Total volume
9
Table 3
Composition of Reaction Mix for Polymerase Chain Reaction Amplification
Component
Volume for one reaction (
µ
L)
Final concentration
Distilled water
19.5
Advantage PCR buffer (10X)
2.5
1X
dNTP mix (10 m
M
each dATP,
0.5
200
µ
M
each
dGTP, dCTP, and dTTP
at neutral pH)
cDNA polymerase mix (50X)
0.5
1X
Total volume
23.0
5.
Immediately transfer the tube from ice to the prewarmed 25°C thermocycler block
for a 5-min incubation. Prewarm the other thermocycler block at 55°C.
6.
Transfer the tube to the prewarmed 55°C thermocycler block for a 1-h incubation.
7.
Heat inactivate at 72°C for 15 min.
8.
Add 1
L (2 U) of RNase H and incubate at 37°C for 20 min to remove RNA
complementary to the cDNA.
µ
9.
Dilute the product two- to fivefold with distilled water. Store at -20°C before use.
3.4. Primary PCR Amplification
1.
Inside a hood dedicated for setting up PCR, assemble the PCR master mix for the
50 reactions according to
Table 3
with cDNA polymerase mix (
see
Note 6)
in a
