Biomedical Engineering Reference
In-Depth Information
PROTOCOL 5.6 Coupling of Monofunctional Reactive Dyes
to Aminoallyl-Containing aRNAs
Equipment and reagents
•
Dimethyl sulfoxide (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 (MessageAmp
™
kit component, Ambion)
•
Coupling buffer (MessageAmp
™
kit component, Ambion).
•
Method
1 For each aRNA sample, resuspend a tube of dye in 3
μ
l of DMSO immediately prior to use.
Vortex to completely redissolve the dye and centrifuge briefly to collect the dye at the
bottom of the tube.
2 To each purified 7
μ
laRNAsample(fromstep12ofProtocol5.5),add3
μ
l of cyanine or
Alexa dye. Add 9
μ
l of coupling buffer. Mix by pipetting up and down, and incubate in
the dark for 30min at room temperature to allow chemical coupling between dye
molecules and aminoallyl groups on the aRNA.
3 After the 30min coupling reaction, quench the reaction by adding 4.5
μ
lof4
M
hydroxylamine and incubating at room temperature for 15 min in the dark.
PROTOCOL 5.7 Purification of Fluorescently Labeled aRNA
Equipment and reagents
•
aRNA binding buffer (MessageAmp
™
kit component, Ambion)
•
ACS-grade 100% (v/v) ethanol
•
aRNA filter cartridge (MessageAmp
™
kit component, Ambion)
•
aRNA collection tube (MessageAmp
™
kit component, Ambion)
•
aRNA wash buffer with ethanol added according to manufacturer's directions
(MessageAmp
™
kit component, Ambion)
•
Nuclease-free water (Sigma) prewarm to 50
◦
C
•
Vacuum evaporator (SpeedVac; ThermoElectron)
•
UV-vis spectrophotometer.
Method
1 Add 73.5
μ
l of nuclease-free water to a dye-labeled aRNA sample (from step 3 of
Protocol 5.6) so that each sample has a total volume of 100
μ
l.
