Found 1 discussion for this experiment
2 years ago
2 years ago by Alex Santos Ribeiro
I have extracted RNA from brain tissue but my RNA concentrations are as low as 5ng/ml with my highest being around 80ng/ml. Will I be able to perform cDNA conversion with concentrations as low as these?
Found 12 matching solutions for this experiment
|Denature the template-primer mixture by heating the tube for 10 min at +65°C.
Incubate the reaction for 10 to 30 min at +45°C to +55°C.
Inactivate Transcriptor High Fidelity Reverse Transcriptase by heating to +85°C for 5 min
|For downstream PCR, use 1 to 5 μl of the reaction product.
The cDNA can be used for amplification without further purification or manipulation (e.g. RNase H-treatment)
|This kit is optimized for RT reactions using a wide range of total RNA amounts (10 pg -2 μg), such that long and low
abundance cDNAs can be detected by amplification after cDNA synthesis.
|Incubate samples at 45 °C for 30 min.
Incubate samples at 45 °C for 30 min
|The use of higher incubation temperatures up to 48 °C may increase the yield of cDNA synthesized in cases of complex RNA secondary structure|
|Heat Oligo mixture to 65°C for 5 min and quick chill on ice and later add reaction components.
Mix and incubate at 42°C for 2 min.
Add 1 μL (200 units) of SuperScript™ II RT and mix by pipetting gently up and down.
Incubate at 42°C for 90 min.
Inactivate the reaction by heating at 85°C for 5 min.
|This Kit is optimized for the
production of targets < 1kb in length.
There will be no loss in functional performance after 20 cycles of freezing on dry ice and thawing on ice
|For downstream reactions, use 1/5th to 1/10th of the first-strand reaction (2-4 μL) for PCR amplification|