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One-Step RT-PCR Kit

User’s Instruction

Introduction


The One-Step RT-PCR Kit is designed for optimal convenience in carrying out highly sensitive and specific RT-PCR in a single tube. One-step RT-PCR is a variation on standard (two-step) RT-PCR in which all components of the RT and PCR are mixed in one tube prior to starting the reactions and in which RT and PCR are thus carried out sequentially in one tube. This approach offers tremendous convenience when applied to analysis of single targets from multiple samples of RNA. This approach also minimizes the possibility for introduction of contaminants into reactions between the RT and PCR steps, since the RT and PCR reactions are carried out sequentially, without opening of reaction tubes between steps.


The kit is composed of high quality reagents. The AMV reverse transcriptase is used for RT, and the optimized V-Taq polymerase is used for PCR. A unique buffer, 10×V-Taq buffer suitable for both the RT and PCR steps and crucial for successful one-step RT-PCR, is included, along with dNTPs mixture and RNasin. DEPC-treated water is also provided. Use of this kit and the accompanying protocol will ensure excellent results.


Package and Components

25 reactions

1.     AMV Reverse Transcriptase (25 U/µl)

5 µl

2.     V-Taq Polymerase( 5 U/µl)

25 µl

3.     10×V-Taq Buffer

50 µl

4.     dNTP Mixture (10 mM each)

25 µl

5.     RNasin (40 U/µl)

7.5 µl

6.     DEPC- treated water

0.5 ml

Procedures


(1)   Add the following components into 0.2 ml or 0.5 ml thin-wall tubes:

Total RNA

0.5~1 µg

Upper primer

10~30 pmol

Lower primer

10~30 pmol

10×RT-PCR Buffer

2 µl

dNTP Mixture (10 mM each)

1 µl

AMV Reverse Transcriptase

5 u

U-Taq polymerase

5 u

RNasin

10 u

DEPC- treated water

up to 20 µl


 (2)   Mix the solution and centrifuge briefly, then incubate for 30 min at 45℃.


 (3)   Stop the reaction by incubating at 94 for 5 min and enter the following PCR cycles for 30~40 times immediately: 9430s, 6045s, 721~3min (this amplification parameter is for reference only). Extend at 72 for 7min finally.


Remarks


1.       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.


2.        Avoid RNase contamination. All vessels, reagents and solutions must be sterile, and all procedures must be carried out with gloves.


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, and reaction temperature (37~56).

(3)         Secondary structure of RNA: increase reaction temperature or use random primer.


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