One-step RT-PCR Kit RT-PCR Quick Master Mix. Instruction Manual. (Code No. PCR-311) For research use only

Transcription

1 One-step RT-PCR Kit RT-PCR Quick Master Mix Instruction Manual (Code No. PCR-311) For research use only TOYOBO CO., LTD. Life Science Department OSAKA JAPAN

2 Content 1. Introduction Product Contents Additional Material Required Protocol Related Protocol Trouble Shooting Related Products Warning This Kit is intended to be used for experimental purposes only. It is not be used for clinical or diagnostic purposes nor is it intended for human use. When using this kit, proceed carefully following common laboratory safety procedures. NOTICE TO PURCHASER: LIMITED LICENSE Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155, 5,407,800, 5,322,770, 5,310,652, and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research. No right under any other patent (such as the patented 5 Nuclease Process claims in US Patents Nos. 5,210,015 and 5,487,972) and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

3 1. Introduction RT-PCR Quick Master Mix, the 2 master mix for RT-PCR, is used the heat-resistant rtth DNA Polymerase derived the Thermus thermophilus HB8 indicating the reverse transcriptase activity in the presence Mn 2+. So the reverse transcription and PCR process in the same system. And it is suitable for high-throughput experiment and less chance of being contaminated. Features 1. Reverse transcription and PCR process in the same system It is in the same system for the reverse transcription and PCR with RT-PCR Quick Master Mix and RNA template. So it reacts continuously and fast, which not only is suitable for high-throughput but also reduces the crossing contamination. 2. High efficient to the RNA template with the complex conformation or GC-rich content. Compared with normal reverse transcriptase, in the RT-PCR Quick Master Mix, the rtth DNA Polymerase is heat-resistant enzyme used for reverse transcription and PCR. So it has high performance to the RNA with the complex comformation or GC-rich content in higher temperature, in which it also improves specificity. 3. Fast hot-start In the RT-PCR Quick Master Mix, It adopts the anti-dna Polymerase antibody, which can suppresses the nonspecific amplification.so the enzyme is deactivated in normal condition and re-activated when being heated. Attention The fidelity is not very high for PCR when Mg2+ instead of Mn2+and there maybe some mutations in the PCR amplification product. So it is not recommended to use this kit for gene cloning. If you require high accuracy sequence, please refer to the two-step RT-PCR kit[revertra-plus-] (Section7,Related Products)

4 2. Product Contents This mix includes the following products. 2x RT-PCR Quick Master Mix Reagents Stored Volume -20 C (or at 4 C for no more than 2 month) 625μl x 2 50mM Mn(OAc) 2-20 C 200μl Nuclease-free Water -20 C 1100μl Positive Control RNA (human G3PDH mrna, 5x10 5 copies/μl) -20 C 50μl Control Primer F (10pmol/μl) -20 C 50μl Control Primer R (10pmol/μl) -20 C 50μl 2x RT-PCR Quick Master Mix It is the 2 concentration of RT-PCR solution contained reaction buffer,dntps,rtth DNA Polymerase and anti-dna polymerase antibody. After add the template RNA, primers, Mn(OAc) 2, Please add the Nuclease-free Water diluting to 1 concentration. After melting, please vortex to make it uniform to use and keep it in -20 C again. Don t be thaw- freeze for more than 10 times. If used little once, it is recommend small amount note to be preserved. If it is used up in short term (no more than 2 months), It also can be stored at -4 C. 50mM Mn(OAc) 2 The Mn 2+, the necessary solution for this mix, add 1/20 volume to the reaction system (final concentration 2.5mM).Please adjust the amount depending on the concentration and types of the RNA template to make better performance. Please store at -20 C Nuclease-free Water The nuclease-free sterile distilled water have been removed the DNase and RNase. If the sterile water treated with DEPC, It maybe effect the activity of the polymerase.

5 Control Primer F, Control Primer R (10pmol/μl) The primers are special to the Glyceraldehyde-3-Phosphate Dehydrogenase (G3PDH or GAPDH). These primers span two exons with approximately 450bp PCR product. The positive control RNA can be used as a positive control in the system of the PCR. The G3PDH gene is a housekeeping gene expressed in various mammalian tissues. It is commonly known that the mrna expression level is constant and is not affected by partial inducers including cytokine and phorbol esters. So the G3PDH can be as an internal reference to total RNA or poly(a) + RNA derived from various tissues not only form human but also form mouse, rat, swine, and so on. The sequence for each primer Control Primer F : 5'-TCCACCACCCTGTTGCTGTA-3' (20mer) Control Primer R : 5'-ACCACAGTCCATGCCATCAC-3' (20mer) G3PDH mrna Primer F 559 intron Primer R Size (bases) The position of the each primer. Positive Control RNA (human G3PDH mrna, 5x10 5 copies/μl) The RNA is an in vitro transcription product derived human,whose 3 end has been added 22mer of poly(a).the Control Primer F,R can be used as a postive control primers in the system of RT-PCR.

6 3. Additional Material Required Please prepare the following reagents and devices when available. Instruments and tubes for PCR Please just follow the instruction when use. Primer In order to get high detective sensitivity, It is very important to design the primer corresponding to target sequence. To avoid the infection of the genomic DNA, the designed primers should span two exons. If the gene special primer is used as reverse primer, it is no need to use Random Primer Oligo(dT) Primer again. Nuclease-free Water This product is used to dilute the template RNA. Please prepare additional if necessary. It is not recommended to use the nuclease-free water treated with DEPC. Please don t share the same nuclease-free water with other experiments to avoid contaminated. Total RNA The total RNA can be used as the template directly. It maybe incorporate the genomic DNA when purified the total RNA used the AGPC (Acid Guanidium-Phenol-Chloroform) method. So it may check out the pseudgene in many cases.if necessary, remove the genomic DNA with DnaseⅠ. The expression analysis proved that it usually includes 1-2% mrna in the total RNA derived organization, culture cells. Poly(A) + RNA (mrna) Poly(A) + RNA can be separated selectively with oligo(dt) or hybridization method. So the concentrated mrna can be detected with the high sensitivity. However, compared to the total RNA, the mrna is degraded easily by the RNase.So please pay more attention when purified..

7 Please prepare the following items when analyzing the results of the RT-PCR. (1)Reagents Electrophoresis gel Electrophoresis buffer DNA maker Loading Dye (2)Instruments Gel electrophoresis device UV trans-illuminator and documentation system Micro-centrifuge 4. Protocol The standard protocol is stated below: (1) Prepare the reaction solution Nuclease-free Water up to 50 μl 2x RT-PCR Quick Master Mix 25 μl 50mM Mn(OAc) μl (final. Conc. 2.5mM) Forward Primer 10 pmol (final. Conc.0.2μM) Reverese Primer 10 pmol (final. Conc.0.2μM) RNA sample <2.5μg Total volume 50 μl The mix used with positive primers and positive control RNA Nuclease-free Water 16.5 μl 2x RT-PCR Quick Master Mix 25 μl 50mM Mn(OAc) μl (final. Conc. 2.5mM) Control Primer F (10pmol/μl) 2 μl (final. Conc. 0.4μM) Control Primer R (10pmol/μl) 2 μl (final. Conc. 0.4μM) Positive Control RNA (5x10 5 copies/μl) 2 μl (10 6 copies) Total volume 50 μl The nuclease-free water is recommended used the attached, self-preparation or commerical RNase-free water. If using the water treated with DEPC which maybe hamper the reaction. Please heat it with autoclave to remove the residual DEPC.

8 Please don t share the same RNase-free water with other experiments. The amount of the primer is 2-6pmol (finl.conc μm) each. Please add the amount if the amplification efficiency is not well and reduce the amount if there are too nonspecific signals. The final concentration of Mn(OAc) 2 is almost 2.5mM. Please increase or decrease the amount depending on the concentration of RNA and target sequence to get a better result. For the amount of RNA specimen,total RNA is less than 2.5μg, poly(a) + RNA is less than 500ng. The additional amount may reduce the efficiency of the reaction. (2) PCR Set RT-PCR temperature. Denature 90 C 30s RT 60 C 30min. Denature 94 C 1min. PCR 94 C 30s (40 cycles) C 30s 72 C 1min. Elongation 72 C 7min. This product contains the hot start polymerase and it is enough for pre-denature with 90 C 30s. The longer heated will reduce the activity of the enzyme and the stability of RNA template. The temperature and the number of the cycles with the PCR can be changed depending on target RNA. It is also possible to use the two-step PCR.

9 5. Related Protocol 1. Total RNA treated with DNase I The total RNA purified with the AGPC method maybe incorporate the genomic DNA. The following is the method to remove the genomic DNA. (1)Preparation of the reaction solution Distilled Water(RNease-free grade) Total RNA 10 DNase I Buffer RNase-free DNase I (10U/μl) Total volume up to 10 μl x μl 1 μl 0.5 μl 10 μl When the reaction solution prepared, please leave on ice for 10-30min. (2) Purification (A commercial purification kit can be used) After preparing reaction solution, please leave on ice for 10-30min. Add Distilled Water up to 100μl, Add 100μl TE saturated phenol, Vortex, leave on ice for 5min. 12,000rpm, 5min, distract the supernatant Add 100μl chloroform to the Mixture 12,000rpm, 5min, distract the supernatant Add 5μl 20mg/ml glycogen, 100μl 5M Ammonium Acetate, 200μl isopropanol to the solution. After mixing, stand at -20 C for 30min. 12,000rpm, 5min, dispose the supernatant Add 70% ethanol to the deposition 12,000rpm,5min,dispose the supernatant Add the moderate amount of distilled water to the deposition

10 2. Poly(A) + RNA Purification The low expression level gene can be detected when the poly(a) + RNA purified from the total RNA. The following is the instruction of the MagExtractor -mrna- (code No. NPK-801) used the magnetic beads bound with oligo(dt) to purified the poly(a) + RNA. (1) Preparation and purification with DNase I MagExtractor -mrna- eluate Total RNA (~100μg) 10 DNase I Buffer RNase-free DNase I (10U/μl) Total volume After preparing the solution, leave on ice for 15min. up to 100 μl x μg 10 μl 1 μl 100 μl Add 400μl of Lysis buffer (containing 2-ME), lightly stir with a vortex, then add 800μl of Binding solution and mix well. (2) Purification Magnetic Beads (250μl) add into 1.5ml tube Add the total RNA solution Mix with vortex (until uniform), leave 10min at the room temperature. B/F separation, dispose the supernatant Add 1ml Washing Solution Mix with a vortex (until uniform) B/F separation, dispose the supernatant Add 1ml Washing Solution Mix with a vortex (until uniform) B/F separation, dispose the supernatant Add 1ml Washing Solution Mix with a vortex (until uniform) B/F separation, dispose the supernatant

11 After spin down, re-extract supernatant Add moderate amount of Elution Solution Mix with a vortex (until uniform) Leave for 2min. at 65 C Mix with a vortex (until uniform) Spin down B/F separation, the Poly(A) + RNA is in the supernatant. The standard protocol of MagExtractor -mrna- is the twice of the above purification.so If you want to have a high degree of purification Poly(A)+,Please purified twice. when checking other details of the purified procedure of MagExtractor -mrna, please refer to the instruction manual.

12 6. Trouble Shooting 1. The target band can t be detected by eletrophoresis. Cause The Purity of the template RNA may be poor or degraded. Prepare template again. Remedy The amount of the template RNA may be insufficient. Increase the amount of the template RNA. PCR condition and Tm Value may not match. The concentration of the primers may be too low. The concentration of the Mn(OAc) 2 may be too low. 2. Several extra bands Check annealing temperature, it is generally most suitable at 5 C lower than Tm. The computational method of Tm value might change by the buffer composing. So the recommend annealing temperature may be adjusted. The concentration of the primers adjust at the range of μM.The efficiency of the reverse transcription may be improved if the concentration of the reverse primer is up to1μm Normally, 2.5mM is appropriate. The efficiency of PCR may be raise if add the amount. Cause The concentration of primer is too high Primers have low specificity. Remedy The normal concentration of primer is 0.2μM. Please reduce in some case. Please redesign the primers. The GC content is appropriate at 40-60%. Verify there no complementary area with the primer sequence and 3 end of the sequence. 3. The band can be detected in the blank. Cause It maybe incorporate the positive sample or PCR product. Remedy Firstly, use a blank sample (or water) to try again. If there is also a band, please change the new reagent.

13 7. Related Products The related reagents with purifying RNA Product name Content Code No. mrna purification kit MagExtractor -mrna- 5rxns NPK-801 Total RNA purification kit MagExtractor -RNA- magnetic stand Magical Trapper The related reagents of 2-step RT-PCR 100rxns 1set Products name Content Code No. ReverTra Dash 100rxns PCR-401 NPK-201 MGS-101 ReverTra -Plus- 20rxns 100rxns PCR-501T PCR-501

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