M-MuLV Reverse Transcriptase

Cat. No.: M2000

Price: $160 for 50000U; $536 for 200000U (negotiable)

Storage conditions: -20℃

Concentration: 200 units/μl
 

Component	M2000
M-MuLV Reverse Transcriptase(200U/μl)	20万U

Product Description

This product uses the M-MuLV reverse transcriptase TRUEscript Hˉ RTase, which is cloned and expressed by gene recombination technology and lacks RNase H activity. The RNase H activity contained in the wild-type M-MuLV can catalyze the degradation of RNA in the DNA/RNA hybrid, so it may degrade the template RNA in the RNA/DNA hybrid during the synthesis reaction of the first chain of cDNA. This enzyme M-MuLV (RNase Hˉ) lacks RNase H activity. Compared with M-MuLV, it has stronger extension ability and stability, and can be used for longer cDNA synthesis and the construction of a high proportion of full-length cDNA libraries.

Scope of application: First-chain cDNA synthesis. Can be used for the detection of low-copy genes.

Features: The length of the synthesized cDNA fragment can reach up to 12 kb.

First-chain cDNA synthesis (taking 20 μl reaction system as an example)

1. Add

Components	Volume
Total RNA/mRNA	50 ng-5 μg/5-500 ng
Oligo(dT)18(0.5 μg /μl)or	1 μl
Random Primer(0.1 μg/μl) or	1 μl
GSP(Gene Specific Primer)	2 pmol
dNTP Mixture (10 mM each)	1 μl
5× RT Buffer	4 μl
RNase Inhibitor(40 units/μl)	0.5 μl
TRUEscript Hˉ RTase	0.8-1μl (见注意事项 4)
RNase free H2O to final volume	20 μl

2. Mix gently

If using Oligo(dT)18 or gene-specific primers (GSP), incubate at 42℃ for 30-50min.

If using Random Primer, incubate at 25℃ for 10 min and 42℃ for 30-50 min.

3. Heat at 65℃ for 15 min (or 85℃ for 5 min) to inactivate TRUEscript Hˉ RTase.

RT-PCR

It is recommended to take 1/10-1/5 volume (2-4 μl) of the reverse transcription product as a PCR template.

Recommended PCR conditions (taking 50 μl reaction system as an example)
 

Components	Volume	Final Concentration
cDNA Template	2 μl	as required
Forward Primer (10 μM)	1 μl	0.2 μM each
Reverse Primer (10 μM)	1 μl	0.2 μM each
10×Taq Buffer (含 Mg2+)	5 μl	1×
2.5 mM dNTPs	4 μl	0.2 mM
Taq DNA Polymerase	0.5 μl	2.5 units
ddH2O to final volume	50 μl	Not applicable

PCR cycle

Notes

1. Avoid RNase contamination.

2. To ensure successful reverse transcription, it is recommended to use high-quality RNA samples.

3. If the RNA template is rich in GC or has a complex secondary structure, you can first add only the RNA template, primers and RNase-free H2O and mix them well, denature at 65℃ for 5 minutes, cool on ice, and add other components after a short centrifugation to continue the reverse transcription step below.

4. TRUEscript Hˉ RTase is very viscous, and the solution is easily adsorbed on the tube wall and the outside of the pipette tip, resulting in loss. Please centrifuge before use and avoid loss of adhesion to the outer wall of the pipette tip. You can use 0.8μl each t