Biomedical Engineering Reference
In-Depth Information
5 Prepare a Probe-Quant G-50 column for removal of uncoupled dye material as
follows:
•
resuspend the resin in the column by vortexing;
•
loosen the cap one-fourth turn and snap off the bottom closure;
•
place the column in a 1.5 ml microcentrifuge tube and centrifuge at 735
g
for
1min.
l
6 Place the column into a fresh 1.5 ml tube and slowly apply 50
μ
l of the sample to the
top center of the resin, being careful not to disturb the resin bed.
7 Centrifuge the column at 735g for 2min. The purified sample is collected at the bottom
of the support tube.
8 Discard the column and store the purified and labeled sample
m
in the dark until use on
the same day, or alternatively store at
−
20
◦
C for a maximum of 10 days.
Notes
j
For paraffin-embedded tissue, 600 ng of test and reference DNA samples should be used. We
experienced that reference DNA prepared from either blood or FFPE 'normal tissue' can give
equally good results.
k
Test and reference DNA can be labeled with either Cy3 or Cy5.
l
Start the timer and microcentrifuge simultaneously to ensure that the total centrifugation time
does not exceed 1 min.
m
It is not necessary to exactly quantify the labeled DNA or the degree of Cy5/Cy3-dCTP
incorporation, because in the data analysis a normalization of the Cy5/Cy3 channels takes
place.
PROTOCOL 1.6 Hybridization
Equipment and reagents
•
Blocking solution:
n
0.1
M
Tris, 50 m
M
ethanolamine, pH 9.0: dissolve 6.055 g of Trizma
base and 7.88 g of Trizma-HCl in 900ml of water. Add 3 ml of ethanolamine
(Sigma-Aldrich Chemie B.V. Zwijndrecht, Netherlands) and mix thoroughly. Adjust the
pH to 9.0 using 6 N HCl. Adjust the final volume to 1 l with water.
•
20
×
SSC, pH 7.0 (e.g. Sigma) and dilutions in water (0.2
×
,0.1
×
and 0.01
×
SSC).
•
20% (w/v) SDS solution: for preparation of 100ml, dissolve 20 g of sodium dodecyl
sulfate in 90ml of water. Adjust the final volume to 100ml.
•
Wash solution: 4
×
SSC, 0.1% (w/v) SDS: 200ml of 20
×
SSC, 10 ml of 10% (w/v) SDS,
adjust the final volume to 1 l with water.
•
Human Cot-1 DNA, 1
μ
g/
μ
l (e.g. Invitrogen).
Yeast tRNA, 100
μ
g/
μ
l (e.g. Invitrogen).
•
