Biomedical Engineering Reference
In-Depth Information
3 Meanwhile, prepare the RT master mix as follows (volumes shown are for one 20
μ
l
reaction):
•
2
μ
lof10
×
first-strand buffer
•
1
μ
l of RNase inhibitor
•
4
μ
lofdNTPmix
•
1
μ
l of reverse transcriptase.
Mix, centrifuge briefly to collect the reagents at the bottom of the tube and place
on ice.
4Add8
μ
l of the RT master mix to each sample from step 2. Mix thoroughly, but gently,
b
and place the tubes at 42
◦
C for 2 h to allow synthesis of cDNA molecules from the mRNA
template.
5 Centrifuge briefly to collect sample to bottom of tube and place tubes on ice.
6 Proceed with second-strand cDNA synthesis according to Protocol 5.2.
Notes
a
It has been our experience that, when working with this kit, using a minimum of 200 ng of
total RNA will improve reliability.
b
Do not vortex.
PROTOCOL 5.2 Second-Strand cDNA Synthesis Using
MessageAmp
™
Kit (Ambion)
Equipment and reagents
•
10
×
second-strand buffer (MessageAmp
™
kit component, Ambion)
•
dNTP mix (MessageAmp
™
kit component, Ambion)
•
DNA polymerase (MessageAmp
™
kit component, Ambion)
•
RNaseH (MessageAmp
™
kit component, Ambion)
•
Nuclease-free water (Sigma)
•
Thermal cycler set at 16
◦
C.
Method
1 Prepare the second-strand master mix in a nuclease-free tube in the order listed
(volumes shown are for a single 100
μ
l reaction):
63
μ
l of nuclease-free water
•
10
μ
lof10
×
second-strand buffer
•
