タイトルまたは説明
M-MuLV Reverse Transcriptase
Cat. No.: M***0
Price: $**0 for ****0U; $**6 for *****0U (negotiable)
Storage conditions: **0℃
Concentration: **0 units/μl
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Component
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M***0
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M-MuLV Reverse Transcriptase(**0U/μl)
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*0万U
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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
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Components
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Volume
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Total RNA/mRNA
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*0 ng*5 μg/****0 ng
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Oligo(dT)*8(0.5 μg /μl)or
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1 μl
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Random Primer(0.1 μg/μl) or
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1 μl
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GSP(Gene Specific Primer)
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2 pmol
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dNTP Mixture (*0 mM each)
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1 μl
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5× RT Buffer
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4 μl
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RNase Inhibitor(*0 units/μl)
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0.5 μl
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TRUEscript Hˉ RTase
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0.**1μl (è§æ³¨æ„事项 4)
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RNase free H2O to final volume
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*0 μl
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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)
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Components
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Volume
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Final Concentration
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cDNA Template
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2 μl
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as required
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Forward Primer (*0 μM)
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1 μl
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0.2 μM each
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Reverse Primer (*0 μM)
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1 μl
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0.2 μM each
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*0×Taq Buffer (å« Mg2+)
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5 μl
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1×
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2.5 mM dNTPs
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4 μl
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0.2 mM
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Taq DNA Polymerase
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0.5 μl
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2.5 units
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ddH2O to final volume
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*0 μl
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Not applicable
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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
