Medino®  Advanced One Step RT-qPCR SYBR Green Kit

ProductMedino®  Advanced One Step RT-qPCR SYBR Green Kit
Catalog No.LM601011
Storage conditionsThis product should be stored at -25~-15℃ for 1 year.

Description

Product description Medino® Advanced One Step RT-qPCR SYBR Green Kit is a kit for fluorescence quantification based on SYBR Green I dye. Using gene-specific primers, reverse transcription and qPCR reactions were completed in one tube without repeated open capping and pipetting procedures, greatly increasing detection efficiency and reducing the risk of contamination. For RNA samples, the kit used heat-resistant Medino® Reverse Transcriptase to efficiently synthesize CDNA along with quantitative amplification by Medino® HotStart Taq DNA Polymerase. In the optimized buffer system, the detection sensitivity of the kit against the highly expressed target can be detected up to 0.1 pg, and the moderately expressed target can be detected up to 1 pg. The kit is also suitable for amplification quantification of DNA samples. The kit can realize different animal and plant samples, cells and microbial cores Highly sensitive detection and quantification of acids.   Components
Cat.No.LM601011
Size20T200T
  Instructions
  1. Recommended reaction system
ComponentsVolume (μL)Volume (μL)Final concentration
2×Medino® Advanced SG Buffer12.525
Medino®  Advanced UH Enzyme Mix2
Forward Primer (10 μM)0.510.2 μM
Reverse Primer (10 μM)0.510.2 μM
Templet RNAXX
RNase-free  ddH₂OUp to 25Up to 50
 
  1. Reaction program
Cycle stepTemp.TimeCycles
Reverse transcription50℃6min1
Predenaturation95℃5min1
Amplified reaction95℃15sec40
60℃30sec
Melt curve phaseInstrument default settings1
 
  1. Applicable model
Instrument model without Rox correction: Bio-Rad:CFX96,CFX384,iCycler iQ,iQ5,MyiQ,MiniOpticon,Opticon,Opticon 2,Chromo4; Eppendorf:Mastercycler ep realplex,realplex2s; Qiagen:Corbett  Rotor-Gene  Q,Rotor-Gene  3000,Rotor-Gene  6000; Roche  Applied   Science:LightCycler  480,LightCycler2.0,Lightcycler   96; Thermo    Scientific:PikoReal Cycler;Cepheid:SmartCycler;llumina:Eco qPCR; Low Rox Applicable models:ABI7500,7500  Fast,ViiA7,QuantStudio  3  and  5,QuantStudio  6,7,12k  Flex; Stratagene MX3000P,MX3005P,MX4000P; High Rox Applicable models:ABI5700,7000,7300,7700,7900HT    Fast,StepOne,StepOne  Plus.  
  1. Primer design techniques
  • Primers should best be designed to span an exon-exon junction, where one of the amplified primers can potentially cross the actual exon-intron boundary. This design Can reduce the risk of amplification to false positives from contaminated genomic DNA.
  • The primers should be specific. After primer design, BLAST should be performed.
  • The primer length is generally from 18 to 27 bp, which should not be too long, otherwise the extension temperature is too high and not suitable for Taq DNA polymerase reaction.
  • The GC content of the primers ranged from 40% to 60%, and either too high or too low GC content was unfavorable to the reaction. The GC content of the upstream and downstream primers should not differ too much. In addition, the Tm value of the upstream and downstream primers is the unwinding temperature of the oligonucleotide, namely the temperature of the 50% oligonucleotide duplex at a certain salt concentration. Effective start temperature, generally higher than And Tm values of 5 to 10℃. The Tm value of the primer can be estimated by the formula Tm=4 (G + C) + 2 (+ A + T), or the primer Tm value can be calculated by software.
  • The length of PCR products is usually controlled at 80 – 300 bp, and to ensure amplification efficiency, the length of PCR products is considered when designing primers.
  • Primer 3 ‘end to avoid as far as possible for A, primer 3’ end mismatch, there is A big difference, when the bottom of the base for A, even in the case of mismatch, also can have the synthesis of the chain, and when the bottom chain T, mismatch cause efficiency is greatly reduced, G, C mismatch cause efficiency between A, T Between, so T at the 3 ′
  • Bases should be randomly distributed without the presence of polypurines or polypyrimidines. In particular, the 3 ′end should not exceed three consecutive G or C, thus making the primer enriched in GC Sequence region error was raised.
  • Avoid the existence of complementary sequences between the primer itself and the primer, otherwise the primer itself would fold into a hairpin structure to renatze the primer itself. This secondary structure can affect the complex binding of the primer to the template due to steric hindrance. The primer itself cannot have four consecutive bases. There should be no complementarity between the two primers, and especially this should be avoided 3 The complementary ends overlap to prevent the formation of the primer dimers. There cannot be four consecutive bases complementary between the primers. Primer dimer and hairpin structure if unavoidable Therefore, the AG value should not be too high (less than 4.5 kcal / mol). Otherwise, it will lead to the production of primer dimer band and reduce the effective concentration of primer The PCR reaction could not proceed normally.
 
  1. Abnormal outcome analysis
  • No Ct, value appears
  1. Inror in collecting fluorescence signal: collect signal at the end of annealing / extension;
  2. Primer degradation: its integrity can be detected by PAGE electrophoresis;
  3. Insufficient amount of template: the sample of unknown concentration should be tested from the stock solution;
  4. Template degradation: avoid the introduction of impurities and repeated freezing and thawing in the sample preparation.
 
  • Large Ct value (Ct> 35)
  1. Low amplification efficiency: the reaction condition is not optimized enough, the primer design has problems, the three-step method can be tried for the reaction, and the annealing temperature can be also appropriately reduced;
  2. The PCR product is too long: the product length of 80-150bp is generally used
 
  • The standard curve has a poor linear relationship
  1. Add sample error: make the standard non-gradient; RNA template is recommended for gradient dilution using 1 TE buffer , DNA template The use of ddH₂O dilution is recommended;
  2. Degradation of standards: standards shall avoid repeated freezing and thawing, or repreparation and dilution of standards;
  3. Poor primer design: redesign of primers;
  4. Presence of inhibitors in the template, or excessive concentration of the template.
 
  • The NTC was amplified (Ct <32)
  1. The primer design is not optimized enough: the appearance of the primer dimer and hairpin structure should be avoided;
  2. Poor concentration of primers: reduce the concentration of primers appropriately, and pay attention to the concentration ratio of upstream and downstream primers;
  3. Aerosol pollution: the leakage of amplification products is easy to cause aerosol pollution in the laboratory. Once the pollution, the quantitative test results will be inaccurate. Recommended use DNA / RNA Remover nucleic acid aerosol contamination cleaner .
  Notes
  1. Samaddition and dispensing steps are operated on ice as far as possible
  2. Each component should be shaken and centrifuged at low speed before use
  3. For your safety and health, please wear a lab coat and disposable gloves during operation.
  4. This product is for research use only
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