Biomedical Engineering Reference
In-Depth Information
Notes
f
The multiplex PCR amplifies three amplicons, one each of of 150 bp, 255 bp and 511 bp. This
method is comparable to the van Beers method [38].
g
Use 10 ng of genomic DNA prepared from freshly frozen tissue as a control.
h
If the DNA concentration (see Protocol 1.9) is higher than 5 ng/
μ
l it can be diluted in water.
i
The 150 and 255 bp amplicons have to amplify for a DNA template considered to be suitable
for aCGH.
PROTOCOL 1.5 Labeling of DNA for Oligonucleotide aCGH
Equipment and reagents
•
BioPrime DNA labeling system (Invitrogen, 18094-011), containing:
—2.5
×
random primers solution
—Klenow fragment of DNA polymerase I (40 U/
μ
l); keep on ice at all times, or preferably
use a
−
20
◦
C labcooler when taking in and out of the freezer.
Cy3-labeled dCTP (e.g. Amersham Biosciences/Perkin Elmer)
•
Cy5-labeled dCTP (e.g. Amersham Biosciences/Perkin Elmer)
•
ProbeQuant G-50 Micro Columns (Amersham Biosciences)
•
dNTP mixture; for 200
μ
lmix:
—4
μ
l of 100m
M
dATP
—4
μ
l of 100m
M
dGTP
—4
μ
l of 100m
M
dTTP
—1
μ
l of 100m
M
dCTP
—2
μ
lof1
M
Tris-HCl, pH 7.6
—0.4
μ
lof0.5
M
EDTA, pH 8.0
—184.6
μ
l of water.
•
Method
1 In a PCR tube, mix 300 ng
j
of genomic DNA and 20
μ
lof2.5
×
Random Primers solution.
Adjust the volume to 42
μ
l with water.
2 Denature the DNA mixture in a PCR machine at 100
◦
C for 10 min and immediately
transfer to an ice/water bath for 2-5 min. Briefly centrifuge and put back on ice.
3 While maintaining on ice, add 5
μ
lofdNTPmixture,2
μ
l of Cy3 (test) or Cy5 (ref)
labeled dCTP
k
and 1
μ
l of Klenow DNA polymerase.
4 Mix well and incubate at 37
◦
C (in PCR machine) for 14 h, and then maintain
at 4
◦
C.
