SmartRTTM Reverse Transcriptase is an engineered MMLV RT that improves the enzyme’s thermostability, reduces RNase H activity and its cDNA synthesis ability. The enzyme also has a terminal transferase activity, where it adds a few extra nucleotides to the end of the synthesized cDNA. With a 3’ modified oligo dT primer and a 5’ SMART universal oligo containing a terminal complementation to nucleic acids at the 3’ end of the first-strand cDNA, the SmartRTTM reverse transcriptase will produce RACE ready full-length cDNA.
General Description SmartRTTM Reverse Transcriptase is an engineered MMLV RT that improves the enzyme’s thermostability, reduces RNase H activity and its cDNA synthesis ability. The enzyme also has a terminal transferase activity, where it adds a few extra nucleotides to the end of the synthesized cDNA. With a 3’ modified oligo dT primer and a 5’ SMART universal oligo containing a terminal complementation to nucleic acids at the 3’ end of the first-strand cDNA, the SmartRTTM reverse transcriptase will produce RACE ready full-length cDNA. FeaturesMCLAB\'s reverse transcriptase is one of transcriptases that has the highest thermostability with terminal transferase activity. SourceE. coli ApplicationsReverse transcription and RACE ready full length cDNA synthesis Supplied with 5 x first strand cDNA synthesis buffer Recommended Storage Condition -20 °C Unit Definition One unit is defined as the amount of enzyme that will incorporate 1 nmol of dTTP into acid-insoluble material in 10 minutes at 37°C using poly(A):oligo(dT) as template:primer. Shipping Conditiondry ice User protocolFor RACE ready first-strand cDNA SynthesisCombine the following in separate 0.2-ml PCR tubes (20 µl reaction):1–9 µl RNA sample1 µl 5’ SMART universal Primer1 µl oligo dT Primer1 µl dNTPAdd sterile H2O to a final volume of 12 µl for each reaction.Incubate the tubes at 65°C for 5 minCool the tubes on ice for 3 minAdd the following to each reaction tube:4 µl 5X First-Strand Buffer1 µl DTT (20 mM)2 µl dNTP Mix (10 mM)2 µl SmartRT reverse transcriptaseIncubate the tubes:50°C for 1.5 hr85°C for 5 minFor regular first-strand cDNA SynthesisCombine the following in separate 0.2-ml PCR tubes (20 µl reaction):1–10 µl RNA sample1 µl oligo dT Primer or Random primer1 µl dNTPAdd sterile H2O to a final volume of 12 µl for each reaction.Incubate the tubes at 65°C for 5 minCool the tubes on ice for 3 minAdd the following to each reaction tube:4 µl 5X First-Strand Buffer1 µl DTT (20 mM)2 µl dNTP Mix (10 mM)2 µl SmartRT reverse transcriptaseIncubate the tubes:50°C for 45 min85°C for 5 min |
