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Two-Step RT-PCR Kit

User’s Instruction

Introduction:

This Two-Step RT-PCR Kit has all the reagents needed to synthesize the first-strand cDNA followed by amplification of the cDNA product using PCR. Oligo(dT)18 and random nonamer primers (dN)9 are provided according to different experimental needs. The Kit uses M-MLV Reverse Transcriptase, which is the protein product of mouse leukemia virus gene pol with a single 71 kDa peptide chain. The enzyme has a low RNase H activity. Using oligo(dT)18 or random nonamer as the primer, M-MLV RTase synthesizes the first-strand cDNA. PCR reagents provided in this kit are then used to amplify the cDNA. This kit provides a complete solution from RNA to PCR products, simple and convenient for use.

Package and Components:

25 reactions

1. M-MLV Reverse Transcriptase (200U/µl)

2500U/12.5 µl

2. 5×M-MLV Buffer

100 µl

3. 100 mM DTT

50 µl

4. dNTPs(10 mM each)

37.5 µl

5. RNasin (40U/µl)

250U/6.25 µl

6. Oligo(dT)18 (20 pmol/µl)

0.5 OD/tube

7. (dN)9 (20 pmol/µl)

0.5 OD/tube

8. DEPC-treated water  

1.5 ml

9. Taq DNA polymerase (5U/µl)

50U/10 µl

10. 10×PCR Buffer

50 µl

Procedures

1.     Reverse transcript reaction

(1)   Mix the followings in a sterile thin-wall microtube:

                        Total RNA                                     0.5~1µg

                        (dN)9 or oligo(dT)18                     100pmol

(2)   Denature the mixture at 70℃ for 5min, and cool the tube on ice rapidly, then add the followings sequentially:

5×M-MLV Buffer

4 µl

dNTPs(10 mM each)

1 µl

RNasin

10 U

100 mM DTT

2 µl

M-MLV Reverse Transcriptase

100 U

DEPC-treated water

up to 20 µl

(3)   Mix the solution and centrifuge briefly, then incubate for 1 hr at the appropriate temperature: 42℃ for oligo(dT)18 primers, and 37for (dN)9 primers.

(4)   Stop the reaction by incubating at 94℃ 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.

2.      PCR amplification

(1)   Mix the followings in a sterile thin-wall microtube:

Synthesized cDNA

1-3 µl

10×PCR Buffer

2 µl

Forward primer

10 pmol

Reverse primer

10 pmol

dNTPs(10 mM each)

0.5 µl

U-Taq DNA Polymerase

1.5 U

ddH2O

up to 20 µl

(2)   Mix the solution and centrifuge briefly, then begin the PCR amplification:

Predenature the template at 94℃ for 2.5min. Then enter the following cycles for 30~40 times (this amplificationparameter is for reference only): 9430s, 60℃45s, 72℃1~3min. Extend at 72℃ for 7min lastly.

Remarks:

¨        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 carried out 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.

Troubleshooting Guide

1.        Why the yield of cDNA is low?

Possible causes: (1) The quality of template RNA was too low. (2) The concentration of RNA was estimated too high. (3) Reverse transcriptase inhibitor existed or reverse transcriptase was insufficient. (4) Reaction volume was too large. The common volume should not be more than 50µl.

2.       Why the long cDNA can’t be synthesized?

(1)RNA has been degraded: all vessels and reagents should be sterile and treated with DEPC to avoid RNase. At the same time, RNase inhibitor should be added into the reverse transcription reaction. (2) Improper reaction condition: condition should be optimized, including quantity of reverse transcriptase, salt concentration, reaction temperature (37~56℃) and concentration of DTT (0.5~10mM). (3) Secondary structure of RNA: increase reaction temperature or use random primer.



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