M-MuLV Reverse Transcriptase

详情

M-MuLV Reverse Transcriptase

Cat. No.: M***0

Price: $**0 for ****0U; $**6 for *****0U (negotiable)

Storage conditions: **0℃

Concentration: **0 units/μl
 

Component	M***0
M-MuLV Reverse Transcriptase(**0U/μl)	*0万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 *2 kb.

First-chain cDNA synthesis (taking *0 μl reaction system as an example)

1. Add

Components	Volume
Total RNA/mRNA	*0 ng*5 μg/****0 ng
Oligo(dT)*8(0.5 μg /μl)or	1 μl
Random Primer(0.1 μg/μl) or	1 μl
GSP(Gene Specific Primer)	2 pmol
dNTP Mixture (*0 mM each)	1 μl
5× RT Buffer	4 μl
RNase Inhibitor(*0 units/μl)	0.5 μl
TRUEscript Hˉ RTase	0.**1μl (见注意事项 4)
RNase free H2O to final volume	*0 μl

2. Mix gently

If using Oligo(dT)*8 or gene-specific primers (GSP), incubate at *2℃ for ****0min.

If using Random Primer, incubate at *5℃ for *0 min and *2℃ for ****0 min.

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

RT-PCR

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

Recommended PCR conditions (taking *0 μl reaction system as an example)
 

Components	Volume	Final Concentration
cDNA Template	2 μl	as required
Forward Primer (*0 μM)	1 μl	0.2 μM each
Reverse Primer (*0 μM)	1 μl	0.2 μM each
*0×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	*0 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 *5℃ 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

国家:	China
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產品組 :	Molecular Biology