#6123 v.02.09 Table of Contents I. Description... 2 II. Kit components... 2 III. Storage... 2 IV. Principle... 3 V. Protocol V-1. Preparation of immobilized mrna... 4 Protocol A: Starting from Tissue or Cells... 4 Protocol B: Starting from Total RNA... 5 V-2. Immobilized cdna Library Construction... 6 VI. Notes... 6 VII. Example PCR reaction using immobilized cdna Library... 7 cdna Synthesis system... Cat. cdna Synthesis Kit...#6120 3'-Full RACE Core Set...#6121 5'-Full RACE Core Set...#6122 PCR related products TaKaRa Ex Taq... #RR001 TaKaRa Taq... #R001 TaKaRa LA Taq...#RR002 Premix Taq (TaKaRa Taq version)... #R004 Premix Taq (TaKaRa Ex Taq version)...#rr003 One Shot LA PCR Mix...#RR004 PCR Amplification Kit... #R011 LA PCR Kit Ver.2.1...#RR013 BcaBEST RNA PCR Kit...#RR023 RNA PCR Kit Ver.2.1... #R019 RNA LA PCR Kit Ver.1.1...#RR012 One Step RNA PCR Kit...#RR024 LA PCR in vitro Cloning Kit...#RR015 LA PCR in vitro Mutagenesis Kit...#RR016 Rapid DNA ligation system DNA Ligation Kit Ver. 1 (rapid ligation system)... #6021 DNA Ligation Kit Ver. 2 (rapid ligation system)... #6022 DNA Blunting Kit...#6025 DNA Extraction Cartridges SUPREC -01 (elution from gel slices)...#9040 SUPREC -02 (concentration & purification)...#9041 TAKARA BIO INC. 1
I. Description Solid phase cdna Synthesis Kit is designed to construct cdna libraries immobilized on magnetic particles directly from tissue, cells or total RNA with a simple single tube procedure. mrna is isolated on oligo (dt) linked-magnetic Porous Glass (MPG ) particles. This mrna-mpg particles are applied to the reverse transcription without further modification using the immobilized oligo (dt) 25 as primers. The entire procedure takes approximately two hours and is completed just in a single tube, which minimizes potential loss of template mrna. Moreover, the procedure is quite simple, no need for phenol extractions or ethanol precipitations. Also the yield of mrna is high as the supplied Magnetic Porous Glass (MPG ) is a porous magnetic bead having large surface area. The immobilized cdna libraries constructed with this kit are stable for several months when stored at 4 C. Each library yields product enough for 20 separate PCR reactions at least. The Immobilized cdna libraries are also ready for use in RT-PCR and substractive hybridization. II. Kit component: [Packaging I] 1. MPG Streptavidin (10 mg/ml)... 7.5 mg 2. Biotinylated Oligo(dT) 25 (0.1 mm)... 7.5 nmol 3. Probe Binding Buffer... 5.0 ml 4. Probe Wash Buffer... 4.0 ml 5. 2x Hybridization Binding Buffer... 26 ml 6. Hybridization Wash Buffer I... 25 ml 7. Hybridazation Wash Buffer II... 12.5 ml 8. Pre-RT Wash Buffer... 2 x 9.5 ml 15. TE Buffer... 5 ml 16. Nuclease-free Water... 1 ml [Package II] 9. 5 x RT Buffer...250 µl 10. AMV Reverse Transcriptase (20 units/µl)...25 µl 11. RNase Inhibitor (20 units/µl)...25 µl 12. dntp Mixture (ea. 2.5 mm each)...200 µl 13. Control Mouse Liver Total RNA (lyophilized)*... 150 µg 14. Control PCR Primer (mouse p53)*... 8 µl *This kit contains Mouse Liver Total RNA (13. in Package II) and Mouse p53 PCR primer set (14. in Package II) for control experiment. Control total RNA is used at Step B-1-1 of V. Protocol in replacement of sample RNA. Performing PCR with this immobilized cdna library from Control Total RNA as template and with p53 PCR primer set can yield the amplified products of 371 bp. III. Storage: Reagents and instruments not included with kit - Nuclease-free dh 2 O for dilution of 2 x Hybridization Buffer - Microcentrifuge - Magnetic stand (for separation of particles) Package I at 4 C Package II at -20 C AMV Reverse Transcriptase is inactivated by repeated freeze-thaw cycles. Therefore, it is recommended to divide the enzyme into 5 µl aliquots and store at -80 C. Once an aliquot is removed from -80 C and thawed, it should be stored at -20 C. AMV Reverse Transcriptase is stable for at least 6 months when stored at 2 TAKARA BIO INC.
IV. Principle A-1. Immobilization of Oligo (dt) 25 on MPG Streptavidin particles MPG Streptavidin prticles Biotinylated Oligo(dT) 25 M SA B : Magnetic particles : Streptavidin : Biotin Oligo(dT) 25 -bound MPG Streptavidin particles A-2. Capture of mrna on Oligo (dt) 25 - bound MPG Streptavidin particles Tissue or Cells AAAAA...AAAAA-mRNA Homogenize in 1x Hybridization Binding Buffer. Add Oligo(dT)25 bound MPG Streptavidin particles into the supernatant (or total RNA). Mix for 5 min. at room temperature to immobilize mrna to Oligo(dT)25 bound MPG Streptavidin particles. AAAAA...AAAAA - mrna Collecting with a magnet, wash the mrna-bound MPG Streptavidin particles in Hybridization Wash Buffer, and then with Pre-RT Wash Buffer. AAAAA...AAAAA - mrna Reverse Transcription AAAAA...AAAAA - mrna - 1st strand cdna TAKARA BIO INC. 3
V. Protocol V-1. Preparation of immobilized mrna Enzymes should be stored at -20 C until use and be returned at -20 C immediately after use. The other kit components are thawed on ice until ready to use. 2x Hybridization Binding Buffer may form precipitates during storage. Warm at 37 C to redissolve the precipitates completely before dilution. Preparation of 1x Hybridization Binding Buffer. (This buffer is required in preparation of immobilized mrna from tissue or cells.) -1 x Hybridization Binding Buffer is prepared by mixing 10 ml of 2x Hybridization Binding Buffer with 10 ml of nuclease-free water (DEPC-treated H 2 O* is recommended.) - Prepared 1x Hybridization Binding Buffer should be stored at -20 C. * DEPC-treated H 2 O is prepared by adding DEPC into sterilized distilled water to a concentration of 0.1%, mixing by stirrer overnight at room temperature or at 37 C, and then autoclaved. For determination of the yield of mrna, see 4 of V. Note. A. Protocol A: Starting from Tissue or Cells Notes: 1. If starting from total RNA, follow Protocol B on page 5. 2. When collecting particles in a magnetic stand is difficult because of high viscosity of the solution, centrifuge for a few seconds prior to being left in a stand. A-1. Immobilization of Oligo (dt) 25 on MPG Streptavidin particles 1. Vortex well MPG Streptavidin particles to fully suspend the particles. Dispense 30 µl (300 µg) of MPG Streptavidin into a 1.5 ml nuclease-free microcentrifuge tube. Set the tubes in a magnetic stand and leave for 1 min quietly. Carefully remove the supernatant. 2. Resuspend the MPG Streptavidin particles in 50 µl of Probe Binding Buffer. Set the tubes in a magnetic stand and leave for 1 min. quietly. Carefully remove the supernatant. Repeat this step two more times. 3. Add 3 µl (300 pmol) of Biotinylated Oligo (dt) 25 Probe and 45 µl of Probe Binding Buffer to each tube. Vortex well and mix using end-over-end rotation for 5 min at room temperature. Set the tubes in a magnetic stand and leave for 1 min quietly. Carefully remove the supernatant. (See 1 of V. Notes.) 4. Resuspend the Oligo (dt) 25 - bound MPG Streptavidin particles in 50 µl of Probe Wash Buffer. Set the tubes in a magnetic stand and leave for 1 min quietly. Carefully remove the supernatant. Repeat this step two more times. 5. Resuspend the Oligo (dt) 25 - bound MPG Streptavidin particles in 50 µl of 1x Hybridization Binding Buffer. A-2. Capture of mrna on Oligo (dt) 25 - bound MPG Streptavidin particles 1. Tissue: Approximately 200 mg of tissue is sufficient for each isolation. Use fresh tissue. To minimize mrna degradation, quickly place the tissue in a 50 ml tube and add enough ice cold 1x Hybridization Binding Buffer to a final concentration of tissue between 0.05-0.10 grams per ml. In case of isolation from plant tissue, the final concentration should be 0.1-0.2 g/ml. Cell: For cells grown as a monolayer: Add 1 ml of ice cold 1x Hybridization Binding Buffer for every 10 6-10 7 cells. Remove cells with a cell scraper and transfer into a 50 ml conical tube. For cells grown in suspension: Pellet cells in a 50 ml conical tube and add 1 ml 1x Hybridization Binding Buffer for every 10 6-10 7 cells. 4 TAKARA BIO INC.
2. Disrupt tissue and cells by homogenizing at 30 second pulses for 1-2 min. with a polytron. For plant tissue, fully grind the tissue using a mortar and pestle sufficiently chilled on a -70 C ice bath or liquid nitrogen or acetone/dry ice. Liquid nitrogen should be added during homogenization to keep a pestle cold enough. Transfer the homogenate into fresh microcentrifuge tubes. (See 2 of 'VI. Notes'. 3. Centrifuge at 14,000 x g for 1-2 min. at 4 C. 4. While centrifugation, leave the tubes containing MPG Streptavidin parcticles in a magnetic stand quietly for 1 min., and then carefully remove the supernatant. 5. After centrifugation, add 1 ml of the supernatant of homogenate into the Oligo (dt) 25 -bound MPG Streptavidin particles prepared at 4. After voltex, mix using end-over-end rotation for 5 min. at room temperature. Leave the tubes in a magnetic stand quitely for 1 min. and remove the supernatant. 6. Wash the mrna-bound MPG Streptavidin particles by resuspending the mrna-bound MPG Streptavidin particles in 0.5 ml of Hybridization Wash Buffer I. Leave the tubes quitely for 1 min. and carefully remove the supernatant. Repeat this step. 7. Wash mrna-bound MPG Streptavidin particles by resuspending in 0.5 ml of Hybridization Wash Buffer II. Leave the tubes quietly for 1 min and careflly remove the supernatant. Wash the particles further by resuspending the particles in 0.25 ml of Pre-RT Wash buffer which has been chilled on ice. Leave the tubes in a magnetic stand quietly for 1 min and carefully remove the supernatant. Repeat this washing two further times. (This washing procedure is essential to completely remove any residual detergent that may remain from the mrna isolation steps.) 8. The prepared mrna-bound MPG particles are ready for construction of immobilized cdna construction. Proceed immediately V-2 Immobilized cdna Library Construction on page 6. Protocol B: Starting from Total RNA B-1 Immobilization of Oligo (dt) 25 on MPG Streptavidin particles 1. Follow the steps 1-4 of A-1 Protocol, page 4. 2. Resuspend the Oligo (dt) 25 - bound MPG Streptavidin particles in 125 µl of 2x Hybridization Binding Buffer, and put it aside until ready for hybridization. B-2 Capture of mrna on Oligo (dt) 25 - bound MPG Streptavidin particles 1. Dispense 75 µg of Total RNA in a 1.5 ml tube. Add nuclease-free H 2 O to bring a total volume up to 125 µl. 2. Heat at 65 C for 2-3 min. to disrupt the secondary structure. 3. Transfer 125 µl of the heated total RNA solution into a tube containing Oligo (dt) 25 - bound MPG Streptavidin particles* resuspended in 2x Hybridization Binding Buffer. (*This is prepared in Immobilization of Oligo (dt) 25 on MPG Streptavidin particles ). Now the final concentration of Hybridization Buffer is 1x. 4. Vortex, and then mix with an end-over-end roter for 5 min. at room temperature. Leave the tubes quietly in a magnetic stand for 1 min. and carefully remove the supernatant. 5. Wash mrna-bound MPG Streptavidin particles by resuspending in 0.5 ml of Hybridization Wash Buffer II. Leave the tubes quietly in a magnetic stand for 1 min. and carefully remove the supernatant. Repeat this washing procedure. 6. Wash mrna-bound MPG Streptavidin particles by resuspending the particles in 0.25 ml of Pre-RT Wash Buffer which has been on ice. Leave the tubes in a magnetic stand quietly for 1 min and carefully remove the supernatant. Repeat this washing two more times. (This washing procedure is essential to completely remove any residual detergent that may remain from the mrna isolation steps.) TAKARA BIO INC. 5
7. The prepared mrna-bound MPG particles are ready for construction of immobilized cdna construction. Proceed immediately V-2. Immobilized cdna Library Construction on page 6. V-2 Immobilized cdna Library Construction VI. NOTES 1. Add the following reagents to the mrna-bound MPG Streptavidin particles. nuclease-free H 2 O 30 µl 5x RT Buffer 10 µl dntp Mixture 8 µl RNase Inhibitor 1 µl AMV Reverse Transcriptase 1 µl 2. Mix gently by pipetting, but make sure that the particles are well suspended. 3. Incubate at 42 C for 1 hour. During this incubation, gently vortex once every 10-15 min. to keep the particles suspended. 4. Leave the tubes in a magnetic stand quietly for 1 min and carefully remove the supernatant. Resuspend the cdna-bound particles in 50 µl of TE Buffer containing 10 mm Tris-HCl, ph8.3, 0.1 mm EDTA. Leave the tubes in a magnetic stand quietly for 1 min. and carefully remove the supernatant. Repeat this washing two more times. *NOTE: Do not heat the immobilized cdna library at or above 42 C prior to PCR. 5. Resuspend the cdna-bound particles in 50 µl of TE Buffer and store at 4 C. The immobilized cdna library is stable for at least 4 months when stored at 4 C.* *NOTE: 1) Do not store the immobilized cdna library frozen. 2) Refer to 5. of V. Notes for control experiment. 1. When capturing of mrna (at the steps of A-2-5. or B-2-4. of V. Protocol), keeping the MPG particles well suspended during incubations greatly improves the efficiency of mrna hybridization to the Oligo (dt) 25 particles. 2. Complete homogenization of tissue and cells is important for maximum mrna capture on MPG particles. In addition, for preparation of full-length mrna, freshly isolated tissue or cells should be immediately disrupted in ice-cold homogenization buffer. It is recommended to homogenize tissue or cells with a polytron homogenizer, or to grind them in liquid nitrogen with a mortar and pestle in case of using plant samples. 3. Each immobilized cdna library can be used for a minimum of 20 reactions of 50 µl PCR. It is possible to adjust the reaction volume depending on an user s requirement. However, too much particles can inhibit PCR reactions. The optimum amount of MPG particles: 15 µg particles / 50 µl PCR The concentrations of immobilized library exceeding 30 µg per 50 µl reaction is inhibitory. 4. To determine the yield of captured mrna, 600 µg of MPG Streptavidin particles is used in A-1. or B-1. of V-1. Protocol. (General protocol uses 300 µg of the particles.) Double the volumes of each component through the steps of A-2-5. or B-2-4. of V-1. Protocol. Resuspend the particles in 500 µl of Hybridization Wash Buffer I and transfer 300 µg (250 µg) into a fresh 1.5 ml tube (Tube 2). Proceed the general protocol with the first tube and synthesize cdna. Wash the particles in Tube 2 twice in Hybridization Wash Buffer II. Remove the supernatant and resuspend in 20 µl of TE Buffer. Elute the mrna by heating at 65 C for 2 min. Place the tubes in a magnetic stand and collect the supernatant containing the eluted mrna. The collected supernatant should contain approximately 1 µg of mrna. The yield and purity can be determined by measuring the optical density (OD) of the isolated mrna at wavelength of 260 nm and 280 nm. Yield of mrna (µg/ml) = OD260 x 44 x dilution rate Purity of mrna = OD260 / OD280 The value of OD260 / OD280 of pure mrna is approximately 2.0 6 TAKARA BIO INC.
VI. Example PCR reaction using immobilized cdna library In case of 50 µl PCR reaction using TaKaRa Ex Taq (#RR001) Reaction mixture: Immobilized cdna library (6 µg/µl) * 2.5 µl 10x Ex Taq Buffer 5 µl dntp Mixture (ea. 2.5 mm) 4 µl Primers ** X µl (final concentration 0.2-1 µm) TaKaRa Ex Taq (5 units/µl) 0.25 µl Sterilized distilled water up to 50 µl * The optimal amount of immobilized cdna library in 50 µl PCR reaction is 15 µg. Amplification efficiency is not necessarily increased by adding more amount of immobilized library to the PCR reaction. Concentrations of immobilized library exceeding 30 µg per 50 µl reaction is inhibitory. **In control experiment, use 4 µl of Control PCR Primers supplied in the kit. PCR condition: In case of control experiment (amplification of 371 bp) 94 C, 5 min. 94 C, 45 sec. 60 C, 45 sec. 68 C, 90 sec. 35 cycles 68 C, 7 min. The optimal PCR condition varies depending on primer sequence, target, and amplified product. Non-specific bands may appear at high molecular weight when Taq is added to a cold reaction mixture. Use of Hot Start method is recommended to eliminate this problem. Other than TaKaRa Ex Taq, the following products are available for PCR from TaKaRa. TaKaRa Taq... (Cat.#R001) TaKaRa LA Taq... (Cat.#RR002) Premix Taq (TaKaRa Taq version)...(cat.#r004) Premix Taq (TaKaRa Ex Taq version).. (Cat.#RR003) One Shot LA PCR Mix... (Cat.#RR004) PCR Amplification Kit... (#R011) LA PCR Kit Ver.2.1...(#RR013) MPG Streptavidin... (#6124A) NOTICE: MPG is a registered trademark of CPG Inc. Magnetic Porous Glass and certain application in which it is used are covered by US patent 5,610,979 and 5,610,274 owned by CPG, Inc. TAKARA BIO INC. 7