Biomedical Engineering Reference
In-Depth Information
4 Combine 1
μ
lof10
×
exonuclease I buffer and 1
μ
l of exonuclease I with 8
μ
lof
nuclease-free water. Add 1
μ
l to the RT reaction mixture. Incubate in the thermal cycler
at 37
◦
C for 15 min, followed by 15min at 80
◦
C.
5 Add 0.7
μ
lof75m
M
MgCl
2
and 0.5
μ
l of RNase H. Mix well and incubate at 37
◦
Cfor
15min.
6 Add 6.5
μ
lof2
×
tailing buffer and 0.7
μ
l of TdT. Mix well and incubate at 37
◦
Cfor
15min. Heat inactivate the TdT by incubating at 65
◦
C for 10min.
Notes
v
In addition to a single cell, this reaction can be performed on 10-1000 pg of total RNA in a
volume of 0.5
μ
l. The final volume of this reaction should be 5
μ
l.
w
It is important to use SuperScript III, as this enzyme has been engineered to be stable at
50
◦
C. The increased temperature used during the RT helps to reduce secondary structure of the
RNA, providing a more reliable representation of the entire mRNA pool.
PROTOCOL 5.9 Second-Strand cDNA Synthesis and PCR
Amplification
Equipment and reagents
•
Recombinant Taq DNA polymerase and included reagents (Taq DNA polymerase, 10
×
Taq
buffer, 50 m
M
MgCl
2
; Life Technologies)
•
SR-T24 primer (5
-GTT AAC TCG AGA ATT CTT TTT TTT TTT TTT TTT TTT TTT T-3
;
425m
M
)
•
4
×
25mM dNTPs (combine equal amounts of each 100 m
M
PCR-grade dNTP; Life
Technologies)
•
75m
M
MgCl
2
•
Nuclease-free water (Sigma)
•
Thermal cycler.
Method
1 Prepare a PCR master mix by combining (for each RT product):
2
μ
lof10
×
Taq buffer
•
0.6
μ
lof50m
M
MgCl
2
•
0.7
μ
lof4
×
25m
M
dNTPs
•
1
μ
lofSR-T24primer(425m
M
)
•
10.7
μ
l of nuclease-free water.
•
