SmartRT™ Reverse Transcriptase Kit

General Description

SmartRT^TM 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 SmartRT^TM reverse transcriptase will produce RACE ready full-length cDNA. 

Features
MCLAB's reverse transcriptase is one of transcriptases that has the highest thermostability with terminal transferase activity.

Source
E. coli

Applications
Reverse 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 Condition
dry ice

User protocol
For RACE ready first-strand cDNA Synthesis
Combine the following in separate 0.2-ml PCR tubes (20 µl reaction):
1–9 µl RNA sample
1 µl 5’ SMART universal Primer
1 µl oligo dT Primer
1 µl dNTP
Add sterile H₂O to a final volume of 12 µl for each reaction.
Incubate the tubes at 65°C for 5 min
Cool the tubes on ice for 3 min
Add the following to each reaction tube:
4 µl 5X First-Strand Buffer
1 µl DTT (20 mM)
2 µl dNTP Mix (10 mM)
2 µl SmartRT reverse transcriptase
Incubate the tubes:
50°C for 1.5 hr
85°C for 5 min

For regular first-strand cDNA Synthesis

Combine the following in separate 0.2-ml PCR tubes (20 µl reaction):
1–10 µl RNA sample
1 µl oligo dT Primer or Random primer
1 µl dNTP
Add sterile H₂O to a final volume of 12 µl for each reaction.
Incubate the tubes at 65°C for 5 min
Cool the tubes on ice for 3 min
Add the following to each reaction tube:
4 µl 5X First-Strand Buffer
1 µl DTT (20 mM)
2 µl dNTP Mix (10 mM)
2 µl SmartRT reverse transcriptase
Incubate the tubes:
50°C for 45 min
85°C for 5 min