cDNA Reaction
Materials and reagents:
iScript Reaction Mix (xxxxxx)
RNase away (Fisher) or equivalent (RNA Zap may be found around the lab – this is
not preferred, as it has a tendency to foam)
200 ul PCR tubes (USA Scientific)
Aerosol resistant filter tips, 20 ul, 200 ul and 1000 ul (various suppliers) – use only
newly opened, pre-sterilized boxes – do not use autoclaved tips
RNase-free water (LifeTechnologies or Ambion)
Reaction set up:
Component
Volume per reaction
5x iScript Reaction Mix
iScript Reverse Transcriptase
RNA template (100fg to 1ug total RNA)
Nuclease-free water
4 ul
Total volume
20 ul
1 ul
x ul
(15-x) ul
Reaction cycle:
Incubate the complete reaction mix (use a thermal cycler):
5 minutes at 25°C
30 minutes at 42°C
5 minutes at 85°C
Hold at 4°C until reactions can be removed.
In addition to the actual samples, standard control reactions are:
iScript Reaction Mix + Reverse Transcriptase + H2O
to check for kit contamination
iScript Reaction Mix + RNA template + H2O
to check for DNA contamination of sample
For quality control and to check for DNA contamination, run a standard PCR reaction on
the cDNA samples, using a primer set known to work, and preferably one to a gene that
would be expressed in the sample conditions. The complete cDNA reactions should have
amplification, while the controls should not.
Note: cDNA does not react well to multiple freeze-thaw cycles, so minimize the number of
times you use a single sample.