cDNA synthesis Yeast

The formation of DNA from an RNA template using reverse transcription leads to the formation of double-stranded complementary DNA or cDNA. The challenges with this process include 1. Maintaining the integrity of RNA, 2. Hairpin loops or other secondary structures formed by single-stranded RNA can also affect cDNA synthesis, and 3. DNA-RNA hybrids, which may result when the first strand of cDNA is formed. For the first challenge, using workflows that involve proper isolation and storage of RNA, and maintaining a nuclease-free environment helps obtain RNA with ideal 260/230 ratios. Using a reverse transcriptase that can tolerate high temperatures (50-55oC), overcomes obstacles imposed by secondary RNA structures. Finally, RNase H has the ability to hydrolyze RNA before the formation of a second cDNA strand. It is important to ensure that RNase H activity is optimal because higher RNase H activity leads to premature degradation of the RNA template. Many reverse transcriptases offer built-in RNase H activity.

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Found 6 matching solutions for this experiment

Upstream tips
Produces high-quality cDNA from nanograms of total or poly A+ RNA.

Store RNA and SMART II A Oligo at –70°C.

Store all other reagents at –20°C
Protocol tips
For ds cDNA Polishing Proteinase K treatment is necessary to inactivate the DNA polymerase activity before proceeding with the ligation steps.

Do not chill the tube at –20°C or on ice before centrifuging. Chilling the sample will result in coprecipitation of impurities
Protocol tips
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 50 min.

Inactivate the reaction by heating at 70°C for 15 min.
Protocol tips
Add 0.5 ul (10 U) of Reverse Transcriptase to the reaction mix and incubate for Incubate 30 min at 55°C.

Inactivate Transcriptor Reverse Transcriptase by heating to 85°C for 5min
Upstream tips
Treat sample with DNase I in the presence of 20 units of RNaseOUT at 37°C for 20 min.

Inactivate DNAse with 25 mM EDTA at 80°C for 2 min
First-Strand cDNA Synthesis Kit

GE Healthcare Life Sciences

Protocol tips
Heat the RNA solution to 65ºC for 10 minutes, then chill on ice.

Add first-strand cDNA reaction mix and incubate at 37°C for 1 hour
iScript cDNA Synthesis Kit

Bio-Rad Laboratories

Protocol tips
Reverse transcribe for 20 min at 46C
Downstream tips
For downstream PCR use only 1/10 of the reaction volume (typically 2ul)
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