MTHFR C677T Mutation Real Time PCR Kit User Manual
Contents
1. Liferiver Revision No ZJO006 Issue Date Jul 1 2012 MTHFR C677T Mutation Real Time PCR Kit User Manual For In Vitro Diagnostic Use Only GD 0135 01 For use with LightC ycler1 0 2 0 Instrument Eo rer Obelis S A Boulevard G n ral Wahis 53 1030 Brussels BELGIUM Tel 32 2 732 59 54 Fax 32 2 732 60 03 E Mail mail obelis net C Vos 1 ual Shanghai ZJ Bio Tech Co Ltd www liferiver com cn Tel 86 21 34680596 trade liferiver com cn Fax 86 21 34680595 2 floor No 15 Building No 188 Xinjunhuan road PuJiang Hi tech Park Shanghai China 1 Intended Use MTHFR C677T Mutation Real Time PCR Kit is used for the detection of methylenetetrahydrofolate reductase MTHFR C677T Mutation in whole blood sample in the real time PCR system 2 Principle of Real Time PCR The principle of the real time detection is based on the fluorogenic 5 nuclease assay During the PCR reaction the DNA polymerase cleaves the probe at the 5 end and separates the reporter dye from the quencher dye only when the probe hybridizes to the target DNA This cleavage results in the fluorescent signal generated by the cleaved reporter dye which is monitored real time by the PCR detection system The PCR cycle at which an increase in the fluorescence signal is detected initially is proportional to the amount of the specific PCR product Monitoring the fluorescence intensities in real time allows the detection of the accumulating produc2. If it is still 28 30 report as 1 For further questions or problems please contact our technical support at trade liferiver com cn B lt 28 T lt 28 T
3. es in reaction Mix DNA extraction buffer is available in the kit and blood samples are used for DNA extraction 4 Kit Contents DNA Extraction Buffer 1 vial 1 8ml MTHER C677T Mutation Reaction Mix 1 vial 450u1 1 vial 12ul 1 vial 400u1 1 vial 30ul 1 vial 30ul PCR Enzyme Mix Molecular Grade Water MTHFR C677T Wild type Positive Control MTHFR C677T Mutant type Positive Control Analysis sensitivity 5 X 10 copies ml Note Analysis sensitivity depends on the sample volume elution volume nucleic acid extraction methods and other factors If you use the DNA extraction buffer in the kit the analysis sensitivity is the same as it declares However when the sample volume is dozens or even hundreds of times greater than elution volume by some concentrating method it can be much higher 5 Storage e All reagents should be stored at 20 C Storage at 4 C is not recommended e All reagents can be used until the expiration date indicated on the kit label e Repeated thawing and freezing gt 3x should be avoided as this may reduce the sensitivity of the assay e Cool all reagents during the working steps e Reaction Mix should be stored in the dark 6 Additionally Required Materials and Devices e Biological cabinet e Real time PCR system e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g e Vortex mixer e Real time PCR reaction tubes plates e Cryo container e Pipets 0 5 ul 1000 ul e Ste
4. for the transport of etiologic agents 9 Procedure 9 1 DNA Extraction DNA extraction buffer is supplied in the kit please thaw the buffer thoroughly and spin down briefly in the centrifuge before use 1 Pipet 50ul non heparin anticoagulation sample to a new 0 5ml tube add 50u DNA extraction buffer close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 2 Incubate the tube for 10 minutes at 100 C 3 Centrifuge the tube at 13000rpm for 10 minutes The supernatant contains DNA extracted and can be used for PCR template Attention A During the incubation make sure the tube is not open Since the vapor will volatilize into the air and may cause contamination if the sample is positive B The extraction sample should be used in 3 hours or stored at 20 C for one month C Different DNA extraction kits are available You may use your own extraction systems or the commercial kit based on the yield For the DNA extraction please comply with the manufacturer s instructions 9 2 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 18 ul 0 4ul Reaction Mix Enzyme Mix 18 4 ul Master Mix 2 ul 18 ul Extraction DNA Master Mix aa an Reaction Plate Tube PCR Instrument 1 The volumes of Reaction Mix and Enzyme Mix per reaction multiply with the number of samples which includes the number of controls standards and sample prepared Molecular Grade Water is used as the negative contr
5. ol For reasons of unprecise pipetting always add an extra virtual sample Mix completely then spin down briefly in a centrifuge Mix completely then spin down briefly in a centrifuge 2 Pipet 18ul Master Mix with micropipets of sterile filter tips to each of the Real time PCR reaction plate tubes Separately add 2ul DNA sample positive and negative controls to different reaction plate tubes Immediately close the plate tubes to avoid contamination 3 Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes 4 Perform the following protocol in the instrument 94 C for 2min 93 C for 5sec 62 C for 30sec 30eveles Fluorescence measured at 62 C y 10 Threshold setting Choose Arithmetic as back ground and none as Noise Band method then adjust the noise band just above the maximum level of molecular grade water and adjust the threshold just under the minimum of the positive control 11 Quality control Negative control and positive control must be performed correctly otherwise the sample results is invalid Wild type positive comol es Mutant type positive contol 235 12 Data Analysis and Interpretation The following results are possible Crossing point value Result Anal O Dam Tsm Resets elow the detection limit or negative lt 28 Blank he sample is of homozygous mutant type TT B lt 28 lt 28 he sample is of heterozygous type CT 28 30 Re test
6. rile filter tips for micro pipets e Sterile microtubes e Disposable gloves powderless e Biohazard waste container e Refrigerator and freezer e Tube racks 7 Warnings and Precaution Carefully read this instruction before starting the procedure e For in vitro diagnostic use only e This assay needs to be carried out by skilled personnel e Clinical samples should be regarded as potentially infectious materials and should be prepared in a laminar flow hood e This assay needs to be run according to Good Laboratory Practice e Do not use the kit after its expiration date e Avoid repeated thawing and freezing of the reagents this may reduce the sensitivity of the test e Once the reagents have been thawed vortex and centrifuge briefly the tubes before use e Prepare quickly the Reaction mix on ice or in the cooling block e Set up two separate working areas 1 Isolation of the RNA DNA and 2 Amplification detection of amplification products e Pipets vials and other working materials should not circulate among working units e Use always sterile pipette tips with filters e Wear separate coats and gloves in each area e Do not pipette by mouth Do not eat drink smoke in laboratory e Avoid aerosols 8 Sample Collection Storage and transport e Collect samples in sterile tubes e Specimens can be extracted immediately or frozen at 20 C to 80 C e Transportation of clinical specimens must comply with local regulations
7. t without having to re open the reaction tube after the amplification 3 Product Description Homozygosity for the T allele of the C677T polymorphism of the gene encoding the folate dependent enzyme 5 10 methylenetetrahydrofolate reductase MTHFR is a risk factor for neural tube defects Both the homozygous TT and heterozygous CT genotypes are associated with lower tissue concentrations of folate higher homocysteine concentrations and lower enzyme activity than the wild type CC genotype these effects are more marked in homozygotes Low folate and raised homocysteine levels in early pregnancy are risk factors for neural tube defects We investigated the possibility that the CT genotype would also increase the risk of these malformations MTHFR C677T mutation detection has a certain guide for anticancer drugs Five fluorouracil for the treatment of TT type patients with better efficacy methotrexate for the TT type patients in the high risk of side effects MTHFR C677T Mutation Real Time PCR Kit contains a specific ready to use system for the detection of MTHRF gene site C677T Mutation by PCR polymerase chain reaction in the real time PCR system One for wild type which labeled by 560nm another for mutant type which labeled by 530nm The master contains reagents and enzymes for the specific amplification of the site C677T Mutation gene DNA Fluorescence is emitted and measured by the real time systems optical unit during PCR There are two prob
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