Product Name :
Detection kit for EGFR Mutations (Fluorescence Probing) Applications :
Detection kit for EGFR Mutations (Fluorescence Probing) Background :
Adopting fluorescence probing technology this kit is suitable for the detection of EGFR six mutation types in serum, plasma or tissue of non small cell lung cancer patients. The detection result can guide the administration of iressa and tarceva, and provide individualized medical basis for non small cell lung cancer patients as well.This kit use TaqMan-MGB probing technology to detect six mutation types. The 5’ end of TaqMan-MGB probe is signed by reporter, for example FAM and VIC, in addition, the 3’ end is non-fluorescence quencher which can’t generate fluorescence and reduce signal background greatly. MGB can enhance hybridization between probe and template, and increase Tm of probe. This shorter probe with MGB can reach higher Tm similarly and make reporter closer to quencher resulting in better quenching effect, lower fluorescence background and higher signal-noise ratio. The probe is complete when it doesn’t bind to template, in addition, the fluorescence generating by reporter is absorbed by quencher, which results in non-fluorescence. Otherwise, the probe is hydrolyzed by 5’→3’ excision enzyme of Taq when it doesn’t bind to template, which makes to separate reporter from quencher and then results in fluorescence. TaqMan-MGB probing method has some advantages such as high sensitivity and specificity. The alternative fluorescence groups of different selectable wavelength can achieve multiplex PCR in one tube, which can reduce the cost of production and improve efficiency. Alternative Name :
1. This kit is only used in vitro detection. 2. This kit should be transported and saved in low temperature. Before using it, please solute adequately and then centrifuge. 3. If the testing sample is tissue, please make sure the tissue includes tumor cells. In addition, if the testing sample is formalin fixed tissue, according to the actual situation loading sample should be more for its degradation. 4. The experiment should be strict zoning operation. The first zone: PCR preparation area-preparing needing reagent for amplification. The second zone: sample handling area-testing sample and reference substance handling The third zone: sample loading and testing area- PCR amplification testing All the things in every zone are special use for avoiding pollution. Please clean the desk after experiment. 5. When splitting reaction liquid, please avoid generating bubble. The samples should be dropped in reaction liquid totally but not adhered to the tube wall when loading samples. After that, please cover the lid quickly. Before on the machine, please make sure all the tubes are closed not to divulge fluorescent substance and pollute machine. 6. Experiment table and all laboratory supplies are sterilized periodically by 1％sodium hypochlorite, 75％ethyl alcohol or ultraviolet lamp. Classification :
Gene Assay Kit Format :
Kit Contents :
In this kit every sample needs three reaction liquids for detection including 19M1(probe 19del 2235-2249, 2236-2250), 19M2(probe 19del 2240-2251, 2240-2257), 21M(probe 2573T＞G、2582T＞A), which signal is indicated by FAM and VIC. The components are shown in table one. Number Components Size Quantity 1 2×Taq Mix 1.2mL/test 1 2 ROX Reference Dye﹡1 48μL/ test 1 3 ROX Reference Dye Ⅱ﹡2 48μL/ test 1 4 19M1 reaction liquid 304μL/ test 1 5 19M2 reaction liquid 304μL/ test 1 6 21M reaction liquid 304μL/ test 1 7 Negative control 20μL/ test 6 8 Positive control 20μL/ test 2 9 product manual 　 1 Storage & Shelf Life :
In this kit 2×Taq Mix and ROX Reference Dye(Ⅱ) can save at four degrees centigrade. The additional reagents can store at twenty degrees below zero. Procedure :
[Self-contained reagents] Tips, PCR 96 pore plates or eight reaction tubes without RNase and DNase. [Suitable instrument] ABI PRISM 7000/7700/7300, StepOnePlus Real-Time PCR System and so on. [Requirement samples] Detectable samples are as follows: 1. Serum, blood, plasma DNA. 2. Tissue including fresh or embedded in paraffin, operation sample conserved in liquid nitrogen, ethyl alcohol and RNAlater. 3. Sample from puncture and biopsy. DNA extraction: Using commercial kit to extract human genomic DNA such as QIAamp DNA Blood Mini Kit. Quantifying extracted DNA and adding less than100ng template in 20μL system. [Calibration method] In every PCR reaction, sample, positive and negative control need analyze simultaneously. 1. Preparing reagent for reaction Taking out nucleic acid reaction solution from the kit and thawing, then centrifuging at 2000rpm for 10sec. 2. Loading samples a). Adding 7.6μL PCR reaction, 10μL 2×Taq Mix and 0.4μL ROX Reference Dye to every tube. Reaction liquid can also be mixed and packaged subsequently. b). Adding 2μL DNA template, and quantifying DNA under 100ng firstly. c). Centrifuging all reagents in PCR 96 pore plates or eight reaction tubes to the bottom. 3. PCR amplification a). Putting PCR reaction plates in fluorescence quantitative PCR instrument. b). Cyclic condition setting