cDNA Synthesis
MasterMixes
HIGHLIGHTS
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Thermostable Reverse Transcriptase blended with Ribonuclease Inhibitor for efficient cDNA synthesis
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Optimized reaction mix with oligo (dT) and random primers for the unbiased representation of mRNA ends
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cDNA synthesis from complex templates at up to 55°C
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High sensitivity detection from 1pg of total RNA template
APPLICATIONS
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cDNA template generation for qRT-PCR or normal PCR
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Unbiased, efficient cDNA synthesis
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Detection of low target template amounts
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cDNA synthesis from complex template
DESCRIPTIONS
MB-cDNA Synthesis MasterMix is highly efficient and easy to use system for cDNA synthesis without reaction optimization. The engineered Reverse Transcriptase ensures high sensitivity detection from even low copy number targets. The Mastermix contains RNase Inhibitor to prevent RNA degreadation thus achieving reaction safety.
The wide reaction temperature range (38°C - 55°C) ensures efficient transcription from GC rich templates. The 5X MB-cDNA sysnthesis MasterMix includes optimal concentrations of magnesium and dNTPs and a combination of anchored oligo (dT) and random hexamers for unbiased representation of mRNA ends.
The kit is an optimal choice for generating high quality cDNA from viral RNA, miRNA or other targets for normal and qRT-PCRs.
MB-cDNA Synthesis MasterMix provides excellent results within the very broad range of the total RNA amount use
4 pg, 40 pg, 400 pg, 4 ng, 40 ng, 400 ng, and 4 μg of mouse total RNA were used for cDNA synthesis under the standard qMB-cDNA Synthesis protocol conditions. An aliquot from each reaction was taken for subsequent qRT-PCR with InView qRT-PCR CYBER Green MasterMix to amplify a 70bp fragment of the mouse RN18S gene. All reactions independently from the initial amount of RNA were100% efficient
PROTOCOL
Tips
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Take care to protect RNA from degradation by keeping your bench clean, wear gloves, use sterile tubes and filter pipet tips
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Include positive and negative controls in parallel
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Thaw and keep reagents on ice. Mix well before use.
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Do not add any other components into the reaction with exception of the template and the supplied reagents.
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The recommended reaction temperature is 42-50°C. For GC rich templates, the temperature can be increased up to 55°C
To Prepare a 20 μl reaction, mix the following:
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5X MB-cDNA Synthsis MasterMix: 4 μl
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Enzyme Blend (20X): 1 μl
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Total RNA: 1 pg to 5 μg
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PCR WaterUp to 20 μl
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Mix gently, avoid bubbles.
Reaction protocol
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Incubate 30 min at 42-50°Cto to synthesize cDNA.
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Inactivate the enzyme 85°C for 10min
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Store reactions at -20°C or on ice for immediate use.
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Use 2-4 μl of this reaction mix per20 μl qRT-PCR reaction