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First Strand cDNA Synthesis
(Cat. # 786‐812)
INTRODUCTION ... 3
ITEMS SUPPLIED ... 3
STORAGE CONDITIONS ... 3
SOURCE ... 3
UNIT DEFINITION ... 3
QUALITY ASSURANCE ... 3
IMPORTANT INFORMATION ... 4
PROTOCOL ... 5
TROUBLESHOOTING ... 6
RELATED PRODUCTS ... 7
INTRODUCTION
First‐strand cDNA Synthesis kit is a complete system for efficient first‐strand cDNA synthesis from total RNA or mRNA. This kit contains oligo(dT)18 and random nonamer primers (dN)9 for different experimental situationss. The kit uses M‐MLV Reverse Transcriptase, which is the protein product of mouse leukemia virus gene pol with a single 71kD peptide chain and exhibits low RNase H activity. Oligo(dT)18 is designed to prime selectively on mRNA with a poly(A)+ tail. A random nonamer can be used for the generation of cDNA libraries, to copy mRNAs that do not contain a poly(A)+ tail, or for the synthesis of the 5' regions of mRNAs. The first‐strand of cDNA can be synthesized using either one of the primers provided or a specific primer of choice.
ITEMS SUPPLIED (Cat. # 786‐812)
Part. # Description Size
127M‐B M‐MLV Reverse Transcriptase [200U/µl] 2,500U
067R‐B RT Reaction Buffer [5X] 100µl
276D‐B DTT [100mM] 50µl
210D‐A dNTPs [10mM each] 25µl
079R‐A Ribonuclease Inhibitor [40U/µl] 250U 346P‐A Primer: Oligo(dT)18 [20pmol/µl] 0.5 OD/tube 345P‐A Primer: (dN)9 [20pmol/µl] 0.5 OD/tube
025D‐E DEPC‐treated water 1.5ml
STORAGE CONDITIONS
The kit is shipped on blue ice. Upon arrival, store at ‐20°C. This product is stable for 1 year at ‐20°C. The M‐MLV Reverse Transcriptase is supplied in 20mM Tris‐HCl, 150mM NaCl, 0.1mM EDTA, 1mM DTT, 0.1% IGEPAL CA‐630, 50% Glycerol, pH7.6.
SOURCE
• M‐MLV Reverse Transcriptase is isolated from an E. coli strain containing a recombinant clone.
UNIT DEFINITION
One unit is defined as the amount of enzyme required to incorporates 1nmol of dTTP into acid‐precipitable material in 10 min at 37°C using poly(A)+ RNA and oligo(dT)18 as template/primer.
QUALITY ASSURANCE
DNase and RNase activity is not detected after incubation of 1μg of DNA and RNA with 200 units of M‐MLV Reverse Transcriptase for 3 hours at 37~42°C.
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IMPORTANT INFORMATION
• Ensure the integrity and purity of your RNA. The quality of RNA is the key for first‐
strand cDNA synthesis. The integrity and purity of RNA can be inspected by the ratio of OD260/OD280 and agarose gel analysis. The common ratio of purified RNA is 1.8~2.0, if not, the RNA should be purified further. The ratio of eukaryotic RNA 28S/18S is about 2:1, if not, the RNA has been degraded.
• Avoid RNase contamination. All vessels, reagents and solutions must be sterile, and all procedures must be performed with gloves.
• Select the right primers for first‐strand cDNA synthesis. Primer oligo (dT)12‐18 can ensure the synthesized cDNAs have intact 3’‐end, and get the first‐strand cDNA close to full length. Primer (dN)6 or (dN)9 can anneal to many sites of the mRNA, and produce short length first‐strand cDNA segments, which is often used to acquire 5’‐end sequence and to obtain cDNA from RNA with secondary structure or stop site that stops the reverse transcription.
• Resuspend the Primer: Oligo(dT)18 in 165µl DEPC treated water to give a 20pmol/µl concentration. Add the water, incubate at room temperature for 5 minutes and pipette up and down to fully dissolve the primer.
• Resuspend the Primer: (dN)9 in 300µl DEPC treated water to give a 20pmol/µl concentration. Add the water, incubate at room temperature for 5 minutes and pipette up and down to fully dissolve the primer.
PROTOCOL
1. Mix 0.5‐1µg Total RNA and 100pmol primer (5µl) ((dN)9 or oligo(dT)18) in a sterile thin‐wall microtube.
2. Denature the mixture in 70°C for 5min, and cool the tube on ice rapidly, then add the following components:
Reagent Volume
RT Reaction Buffer [5X] 4µl
dNTPs [10mM each] 1µl
Ribonuclease Inhibitor [40U/µl] 0.25µl
DTT [100mM] 2µl
M‐MLV Reverse Transcriptase [200U/µl] 0.5µl
DEPC‐treated water up to 20µl
3. Mix the solution and centrifuge briefly, then incubate for 1hr at the appropriate temperature: 42°C for using oligo(dT)18 primers, and 37°C for that using (dN)9
primers.
4. Stop the reaction by incubating at 94°C for 5 min and cool the tube on ice.
5. The cDNA synthesized using this system can be used directly in PCR amplification or other downstream applications.
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TROUBLESHOOTING
Issue Possible Cause Suggestion
Low yield of cDNA
Quality of template RNA was too low
See Important Information for RNA Purity & Integrity
Concentration of RNA was too high
Assay RNA concentration and dilute.
Use 0.5‐1µg.
Reverse transcriptase inhibitor existed or reverse transcriptase was insufficient
See Important Information for RNA Purity & Integrity
Reaction volume was too large Maximum volume should be 50µl
Limited full length cDNA
RNA has been degraded
Ensure all equipment, pipettes and gloves are RNase free. Ensure Ribonuclease Inhibitor is in the reaction.
Poor reaction conditions
Optimize the quantity of reverse transcriptase, DTT concentration (0.5‐
10mM) and reaction temperature (37‐56°C)
Inhibited by RNA secondary structure
Increase reaction temperature or use a random primer
RELATED PRODUCTS
Download our Molecular Biology Handbook.
http://info.gbiosciences.com/complete‐molecular‐biology‐handbook
For other related products, visit our website at www.GBiosciences.com or contact us.
Last saved: 10/9/2012 CMH
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