First Strand cdna Synthesis

Transcription

1 380PR 01 G-Biosciences A Geno Technology, Inc. (USA) brand name First Strand cdna Synthesis (Cat. # ) think proteins! think G-Biosciences

2 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 Page 2 of 8

3 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. # ) 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. Page 3 of 8

4 IMPORTANT INFORMATION Ensure the integrity and purity of your RNA. The quality of RNA is the key for firststrand cdna synthesis. The integrity and purity of RNA can be inspected by the ratio of OD 260 /OD 280 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) 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. Page 4 of 8

5 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. Page 5 of 8

6 TROUBLESHOOTING Issue Possible Cause Suggestion Low yield of cdna Quality of template RNA was too low Concentration of RNA was too high Reverse transcriptase inhibitor existed or reverse transcriptase was insufficient See Important Information for RNA Purity & Integrity Assay RNA concentration and dilute. Use 0.5 1µg. 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 Poor reaction conditions Inhibited by RNA secondary structure Ensure all equipment, pipettes and gloves are RNase free. Ensure Ribonuclease Inhibitor is in the reaction. Optimize the quantity of reverse transcriptase, DTT concentration (0.5 10mM) and reaction temperature (37 56 C) Increase reaction temperature or use a random primer Page 6 of 8

7 RELATED PRODUCTS Download our Molecular Biology Handbook. molecular biology handbook For other related products, visit our website at or contact us. Last saved: 10/9/2012 CMH Page 7 of 8

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