SmartRT™ Reverse Transcriptase Kit

描述

General Description

SmartRT^TM Reverse Transcriptase is an engineered MMLV RT that improves the thermostability of the enzyme, 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.  Together with a 3’ modified oligo dT primer and a 5’  SMART universal oligo, which contains a terminal complementation to nucleic acids at the 3’  end of the first-strand cDNA, the SmartRT^TM reverse transcriptase produces RACE ready full-length cDNA.

Feature
MCLAB’s reverse transcriptase is one of the transcriptases that has a terminal transferase activity with the highest thermostability.

Source
E. coli

Application
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

其他信息

Manual & Protocols

Figure 1.Smart cDNA synthesis compared to conventional cDNA synthesis. Unlike conventional cDNA synthesis methods which involve a multiple enzyme/multiple step procedure, the Smart cDNA synthesis protocol is performed by on reverse transcription reaction, in a single tube, with no adaptor ligation or intervening purification steps. Following PCR amplification, Smart cDNA is immediately available for a variety of downstream applications.