Biomedical Engineering Reference
In-Depth Information
17m
M
sodium bicarbonate, pH 9.0
•
Vacuum evaporator (SpeedVac, ThermoElectron).
•
Method
1 Add 500
μ
l of capture buffer (GFX
™
purification kit) to a GFX
™
column.
2 Transfer a labeled double-stranded DNA PCR product (from step 3 of Protocol 5.12) to
the column and pipette up and down several times to mix the DNA with the capture
buffer.
3 Centrifuge the column at 13 800
g
for 30 s and discard the flow-through.
4 Add 600
μ
l of 80% (v/v) ethanol and centrifuge at 13 800
g
for 30 s, discarding the
flow-through.
5 Repeat step 4 twice, for a total of three times.
6 Centrifuge the column at 13 800
g
for an additional 30 s to ensure that all the ethanol is
removed.
7 Transfer the GFX column to a fresh tube and add 60
μ
lof17m
M
sodium bicarbonate,
pH 9.
cc
8 Incubate the GFX column at room temperature for 1 min.
9 Centrifuge at 13 800
g
for 1min to elute purified, labeled double-stranded DNA.
dd
10 Evaporate the double-stranded DNA sample to complete dryness using a vacuum
centrifugation on high-heat setting, taking care not to over dry the sample. Resuspend
the purified sample in 7
μ
l of nuclease-free water.
Notes
cc
It is crucial that the elution buffer covers the membrane so that when the purified DNA is dried
down, resuspended in 7
μ
l water, and added to 3
μ
l dye/DMSO, the final sodium bicarbonate
concentration is 0.1
M
in the 10
μ
l dye conjugation reaction.
dd
If you wish to stop the protocol at any point and resume the next day you can do so after this
purification step. Simply freeze the aminoallyl double-stranded DNA at
−
20
◦
C. The material is
stable for several days.
PROTOCOL 5.12 Coupling of Monofunctional Reactive Dyes
to Aminoallyl-Containing cDNA
Equipment and reagents
•
DMSO
•
Cyanine 5- and cyanine 3-NHS ester dyes (Enzo Life Sciences)
or
Alexa 647 and Alexa 555
monofunctional reactive dyes (Invitrogen) (dyes are individually packaged in vials for
single reactions)
4
M
hydroxylamine.
•
