Quant One Step RT-PCR Kit
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1 1. Quant One Step RT-PCR Kit For fast and sensitive one-step RT-PCR
2 RT Quant One Step RT-PCR Kit Kit Contents Cat. no. KR113 Contents Hotmaster Taq Polymerase (2.5 U/μl) Quant RTase (for one step) RNasin (40 U/μl) 10 RT-PCR Buffer Super Pure dntp (10 mm each) RNase-Free ddh 2 O 5x RT-PCR Enhancer KR rxn 130 μl 30 μl 30 μl 300 μl 120 μl 2 1 ml 600 μl Handbook 1 Storage Quant One Step RT-PCR Kit should be stored at -20 C. Introduction RT-PCR is the technology by which RNA template is reversely transcribed to cdna by the transcriptase and then target fragment is obtained by amplification using PCR based on the cdna template. RT-PCR is applied in detection of level of genomics in cells, tissues, virus and cloning the cdna of the specific gene. The Quant One Step RT-PCR Kit contains a specially formulated enzyme blend for both reverse transcription and PCR. Only one reaction mix needs to be set up: no additional reagents need to be added after the reaction starts, which avoids contaminations and enhances sensitivity of detection. Although all of the enzymes are present in the reaction mix, the use Quant One Step RT-PCR Kit Handbook 1
3 of Hotmaster Taq polymerase ensures the temporal separation of reverse transcription and PCR, allowing both processes to be performed sequentially in a single tube. Quant One Step RT-PCR Kit contains unique reaction buffers which ensure the high enzymatic activity of the Quant RTase and Hotmaster Taq polymerase. Materials required but not supplied 1. RNase-free ddh 2 O without nucleases 2. RNA templates 3. gene-specific PCR primers Important Notes 1. RNA template could be total RNA or mrna. TRNzol A+ or RNAprep pure kits can be used to purify high-quality total RNA. 2. For some low abundance gene and complex gene, addition of 0.5 μm oligo (dt) may lead to high amplification efficiency. 3. For high-gc and low abundance gene or complex gene, adjusting the reverse transcription temperature may lead to high amplification efficiency. 4. RNase contaminations should be avoided in one-step RT-PCR. Some measures can be taken as below: 1) Wear a disposable gloves and respirator to avoid the RNase contaminations from skin and saliva 2) Operate the RNA related experiments in an RNase-free environment using RNase-free apparatus and consumable items. 3) Consumable items related with RT-PCR should be incubated in 0.1% DEPC solution at 37 C for 12 hours and sterilized for 30 min before use. 5. Quant RTase, RNasin, and Hotmaster Taq polymerase should be centrifuged briefly before use. Pipet slowly and store at -20 C immediately after use. Quant One Step RT-PCR Kit Handbook 2
4 6. Repeated freezing and thawing of dntp should be avoided, since this leads to losing effectiveness 7. Only use gene-specific primers and use specific primers according to specific requirements of experiments. 8. Make sure the thermal cycler is preheated to 50 C before placing samples in it. 9. The primers affect RT-PCR result. GC abundance, primer binding site and length of primers should be considered. Protocol Professional software is recommended for primer design. 1. Thoroughly thaw the template RNA, primer solutions, Super Pure dntp (10 mm each), 10 RT-PCR Buffer, RNase-Free ddh 2 O and 5 RT-PCR enhancer, centrifuge briefly and place them on ice. 2. Prepare a reaction solution according to the following table Contents Volume/Reaction 10 RT-PCR Buffer 5 µl Super Pure dntp (10 mm each) 2 µl 5 RT-PCR enhancer 10 µl RNasin (40 U/µl) 0.5 µl Hotmaster Taq polymerase (2.5 U/µl) 2.5 µl Quant RTase (for one step) 0.5 µl Forward Primer (10 µm) 3 µl Reverse Primer (10 µm) 3 µl Template RNA 10 ng-1 µg total RNA RNase-Free ddh 2 O Up to 50 µl Notes: If setting up more than one RT-PCR reaction, mix all Quant One Step RT-PCR Kit Handbook 3
5 components one time and divide into each tube. 3. Start the RT-PCR program while PCR tubes are still on ice. Wait until the thermal cycler has reached 50 C. Then place the PCR tubes in the thermal cycler. 4. Set up thermal cycler conditions according the following table. Steps Reaction Time Temperature 1 Reverse transcription 30 min 50 C 2 Initial denaturation 2 min 94 C 3 Denaturation min 94 C 4 Annealing min C 5 Extension min 65 C cycles from step 3 to step 5 7 Final extension 10 min 65 C 5. Analyze the PCR products using agarose gel electrophoresis. Quant One Step RT-PCR Kit Handbook 4
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