Biomedical Engineering Reference
In-Depth Information
•
100
×
acetylated bovine serum albumin (BSA) (dilute ultrapure acetylated BSA (Life
Technologies) to 10mg/ml)
•
Reverse transcriptase and included reagents (200 U/
μ
l SuperScript III, 0.1
M
dithiothreitol (DTT); Life Technologies)
•
SR-T24 primer (5
-GTT AAC TCG AGA ATT CTT TTT TTT TTT TTT TTT TTT TTTT-3
,
90.4m
M
)
•
4
×
25m
M
dNTPs (combine equal amounts of each 100m
M
PCR-grade dNTP;
Invitrogen)
•
RNase H (Life Technologies)
•
Exonuclease I and 10
×
exonuclease I buffer (Fermentas)
•
75m
M
MgCl
2
•
Terminal deoxynucleotidyl transferase (TdT) and included reagents (25 m
M
CoCl
2
,5
×
TdT
buffer; Roche)
•
Nuclease-free water (Sigma)
2
×
tailing buffer (80
μ
lof5
×
TdT buffer, 3
μ
l of 100m
M
dATP, 24
μ
lof25m
M
CoCl
2
,
93
μ
l of nuclease-free water)
•
Thermal cycler.
•
Method
1 Prepare first-strand buffer by combining:
•
94
μ
l of lysis buffer
•
5
μ
l of SUPERase-In
•
0.43
μ
lof4
×
25m
M
dNTPs
•
1
μ
l of 100
×
acetylated BSA
•
1
μ
lof0.1
M
DTT
•
0.5
μ
l of 3.68 mM SR-T24.
2 In a 0.2ml microcentrifuge tube, combine the following reagents:
•
single cell
v
•
4
μ
l of first-strand buffer
•
0.5
μ
l of SuperScript III.
w
3 Run the following PCR program:
65
◦
C, 1 min 30 s
•
50
◦
C, 5 min
•
70
◦
C, 10min.
•
