Biomedical Engineering Reference
In-Depth Information
•
2m
M
dNTP mix (1
μ
l of 100m
M
dGTP, 1
μ
l of 100m
M
dCTP, 1
μ
l of 100m
M
dATP and 47
μ
l
of nuclease-free water) (100m
M
PCR-grade dNTPs; Life Technologies)
•
1m
M
dTTP (dilute from 100 m
M
PCR-grade dNTP; Life Technologies)
•
2m
M
aminoallyl-dUTP (dilute from 50m
M
stock; Enzo Life Sciences)
•
Nuclease-free water (Sigma)
Thermal cycler.
•
Method
1 Prepare a PCR master mix by combining (for each PCR product):
•
10
μ
lof10
×
Taq buffer
•
10
μ
lof2m
M
dNTP mix
•
10
μ
lof1m
M
dTTP
•
10
μ
lof2m
M
aminoallyl-dUTP
•
1
μ
lofSR-T24primer(7
μ
M
)
•
3
μ
lof50m
M
MgCl
2
•
0.5
μ
l of Taq DNA polymerase
•
54.5
μ
l of nuclease-free water.
2Dilute1
μ
l of amplified double-stranded DNA PCR product (from step 5 of Protocol 5.11)
1 : 100 in nuclease-free water.
3 In a new 0.2 ml microcentrifuge tube, mix 1
μ
l of the diluted double-stranded DNA PCR
product with 99
μ
l of PCR master mix. Incubate for 25 cycles of:
94
◦
C, 15 s
bb
•
60
◦
C, 30 s
•
72
◦
C, 1min.
•
Notes
bb
Incubate reactions at 94
◦
C for 2 min before beginning PCR amplification if the double-stranded
DNA PCR product has been stored at
−
20
◦
C prior to beginning aminoallyl incorporation.
PROTOCOL 5.11 Aminoallyl Double-Stranded DNA Purification
Equipment and reagents
Illustra
™
CyScribe
™
GFX
™
purification kit (GE Healthcare Life Sciences)
•
80% (v/v) ACS-grade ethanol
•
