A RNase H- reverse trancriptase with improved thermal stability, efficiency and processivity for the production of cDNA from long and difficult mRNA templates.
SunScript™ Reverse Transcriptase RNaseH- is a engineered reverse transcriptase based on the well characterized HIV-1 RT with high thermostability and no Rnase H activity. It has RNA- and DNA-dependent polymerase activity. This enzyme is active up to 85°C. The elimination of RNase H activity enables this enzyme to produce high yields of full length cDNA from long RNA molecules, and the high thermal stability promotes reverse transription through complex secondary structures.
100 ng of total RNA from HeLa cells were reverse transcribed at the indicated temperatures for 60 min with SunScript Reverse Transcriptases and Oligo(dT)18 primer, followed by inactivation at 95°C for 10 min. Presence of GAPDH cDNA was detected by PCR (product length 305 bp).
High reaction temperatures are advantageous to transcribe RNAs with complicated secondary structures.
Sensitivity test: RT reactions were performed at 70°C for 60 min. Inactivation at 95°C for 10 min.
Primer set : Human GAPDH305bp set
1- 1 µg Human total RNA from HeLa cells
2- 100 ng Human total RNA from HeLa cells
3- 10 ng Human total RNA from HeLa cells
4- 1 ng Human total RNA from HeLa cells
5- 100 pg Human total RNA from HeLa cells
1- SunScript™ RT RNaseH+ replicate 1
2- SunScript™ RT RNaseH+ replicate 2
3- SunScript™ RT RNaseH+ replicate 3
4- SunScript™ RT RNaseH- replicate 1
5- SunScript™ RT RNaseH- replicate 2
6- SunScript™ RT RNaseH- replicate 3
Assay for long mRNA reverse transcription: 3 independent RT reactions were performed at 65°C for 60 min using Oligo(dT)20 primers. Inactivation at 95°C for 10 min. PCR Primer sets : mmNEB (>16 kb from 3’end of mRNA; mRNA total length 22,5 kb); mmDnah8 (> 8,5kb from 3’end of mRNA; mRNA total length 14,5 kb)
1 µg of total RNA from HeLa cells was reverse transcribed at 65°C for the indicated times with SunScript™ Reverse Transcriptases and Oligo(dT)18 primer, followed by inactivation at 95°C 10 min.
PCR Primer set: hsHERC4033 (>11 kb from 3’end of mRNA; mRNA total length 15.3 kb)
SunScript™ Reverse Transcriptase RNaseH- is tested to be free of endonucleases, exonucleases and ribonucleases. The purity of the enzyme is guaranteed to be higher than 95%. The enzyme and reagents have been tested for amplification of 16 kb first strand cDNA for 60 min at 65°C.
Each batch of SunScript™ Reverse Transcriptase RNase H- is tested against predetermined specifications to ensure consistent product quality.
Shipping and Storage:
This product is shipped in dry ice. Upon receipt, it should be stored immediately at -20°C in a non-frost-free (constant temperature) freezer. If stored correctly, the product can be kept for at least six months after shipping without displaying any reduction in performance. For longer periods of time, store the kit at -80°C.