Biomedical Engineering Reference
In-Depth Information
Method
1 Prepare an IVT master mix (for each cDNA sample) by combining (at room temperature)
the following reagents in this order:
•
3
μ
l of aa-UTP (50m
M
)
•
12
μ
l of ATP, CTP, GTP Mix (25 m
M
)
•
3
μ
lofUTPsolution(50m
M
)
•
4
μ
lofT710
×
reaction buffer
•
4
μ
lofT7enzymemix.
2 Mix gently by pipetting and centrifuge briefly to collect the reaction mixture at the
bottom of the tube.
3 Transfer 26
μ
l of the IVT master mix to a 14
μ
l cDNA sample (prepared in Protocol 5.3).
Mix thoroughly by flicking the tube and then centrifuge briefly to collect the reaction
mixture at the bottom of the tube.
4 Incubate the reaction at 37
◦
C for 14 h in an air incubator.
j
5Add2
μ
l of DNase I to each reaction. Gently mix and centrifuge briefly to collect the
reaction mixture at the bottom of each tube.
k
6 Incubate at 37
◦
C for 30 min.
7 Add 58
μ
l of nuclease-free water to each aRNA sample to adjust the final volume to
100
μ
l, and mix each reaction mixture thoroughly, but gently.
l
Notes
j
It is important to use an air incubator to prevent condensation formation in the tube cap.
Formation of condensation would alter the concentration of reactants and could result in a
reduced yield.
k
Although the Ambion protocol suggests this is an optional step, we recommend always
performing it as it improves reproducibility.
l
aRNA samples are stable for several days at
−
20
◦
C, providing a convenient stopping place for
users wishing to pause and resume subsequent steps at a later time.
PROTOCOL 5.5 aRNA Purification Using MessageAmp
™
Kit
(Ambion)
Equipment and reagents
•
aRNA binding buffer (MessageAmp
™
kit component, Ambion)
•
ACS-grade 100% (v/v) ethanol
aRNA filter cartridge (MessageAmp
™
kit component, Ambion)
•
