PCR FAQ 1. Nhema dzenhema, hapana bhendi rekusimudzira rinoonekwa 2. False positive 3. Mabhendi ekusimudzira asina-chaiyo anooneka

1Nhema dzisina kunaka, hapana bhendi rakasimudzwa rinoonekwa Iwo akakosha maficha ePCR maitiro ndeaya

① kugadzirira kwetemplate nucleic acid

② primer mhando uye hunhu

③ enzyme mhando uye

④ PCR kutenderera mamiriro. Kuti uwane zvikonzero, ongororo uye tsvagiridzo inofanirawo kuitwa pane zviri pamusoro apa link.

Template:

① Iyo template ine mapuroteni asina kuchena

② Iyo template ine Taq enzyme inhibitors

③ Mapuroteni ari mutemplate haana kugaya uye kubviswa, kunyanya histones mumakromosomes.

④ Yakawandisa yakarasika panguva yekudhirowa nekugadzirira kwetemplate, kana phenol yakafemerwa.

⑤Iyo template nucleic acid haina kubviswa zvachose. Kana kunaka kwema enzymes uye maprimers akanaka, kana mabhendi ekusimudza akasaoneka, zvinowanzoitika kuti pane chimwe chinhu chisina kunaka negayiro remuenzaniso kana template nucleic acid yekubvisa maitiro. Nokudaro, chirongwa chekudya chinoshanda uye chakagadzikana chinofanira kugadzirirwa, uye nzira yacho inofanira kugadziriswa uye haifaniri kuchinjwa pamadiro. . Enzyme inactivation: Izvo zvinodiwa kutsiva iyo enzyme itsva, kana kushandisa ese maenzayimu ekare uye matsva panguva imwe chete kuongorora kana zvisiri izvo zvisiri izvo zvinokonzerwa nekurasikirwa kana kusakwana kwekuita kwe enzyme. Zvinofanira kucherechedzwa kuti dzimwe nguva Taq enzyme inokanganwika.Primers: Primer quality, primer concentration, uye kana kusungirirwa kwemaprimers maviri ari symmetrical zvikonzero zvinowanzoitika zvePCR kukundikana kana kusagutsikana kwekusimudzira mabhendi uye nyore kupararira. Kune matambudziko nemhando yeprimer synthesis mune mamwe mabhechi. Imwe yeaya maprimers ane yakakwira concentration uye imwe ine yakaderera yekumisikidza, zvichikonzera yakaderera-inoshanda asymmetric amplification.

Matanho ekupikisa ndeaya:

① Sarudza yakanaka primer synthesis unit.

② Kuwanda kwemaprimers hakufanirwe kungotarisa kukosha kweOD, asiwo kuterera kune primer stock solution yeagarose gel electrophoresis. Panofanira kuva nemabhandi ekutanga, uye kupenya kwemabhandi maviri ekutanga kunofanirwa kunge kwakafanana. Semuenzaniso, imwe yekutanga ine bhendi uye imwe yekutanga haina bhendi. Kune mitsetse, PCR inogona kukundikana panguva ino uye inofanirwa kugadziriswa kuburikidza nekutaurirana neiyo primer synthesis unit. Kana imwe primer ine kupenya kwakanyanya uye imwe iine kupenya kwakaderera, dzikamisa maratidziro paunenge uchidzikisa maprimers.

③ Maprimers anofanirwa kuchengetwa muhuwandu hwakawanda uye huwandu hudiki kudzivirira kutonhora kudzokororwa uye kunyungudika kana kuchengetedza kwenguva refu mufiriji, izvo zvinogona kutungamirira mukuipa uye kushatisa kwekutanga.

④The primer design haina musoro, sekuti urefu hwekutanga hauna kukwana, dimers inoumbwa pakati pekutanga, etc. Mg2 + concentration: Mg2 + ion concentration ine simba guru paPCR amplification performance. Kana iyo yekumisikidza yakanyanya kuwanda, inogona kuderedza iyo chaiyo yePCR amplification. Kana iyo yekumisikidza yakadzikira, ichakanganisa PCR yekusimudzira goho uye kunyange kukonzera PCR kukwidziridzwa kukundikana pasina mabhendi ekukudza. Shanduko muhuwandu hwekuita: Kazhinji mavhoriyamu anoshandiswa PCR amplification ari 20ul, 30ul, uye 50ul. Kana kuti 100ul, vhoriyamu ipi inofanira kushandiswa kukwidziridzwa kwePCR inoiswa zvinoenderana nezvinangwa zvakasiyana zvekutsvagisa kwesainzi uye kuongororwa kwekiriniki. Mushure mekuita vhoriyamu shoma, yakadai se20ul, uye wobva waita vhoriyamu yakakura, unofanirwa kutevera mamiriro, kana zvisina kudaro ichakundikana nyore. Zvikonzero zvemuviri: Denaturation yakakosha kune PCR kukwidziridzwa. Kana denaturation tembiricha yakaderera uye denaturation nguva ipfupi, nhema zvakaipa zvingangoitika zvikuru kuitika; iyo annealing tembiricha yakadzikira, izvo zvinogona kukonzera kusiri-chaiyo amplification uye kuderedza chaiyo amplification kunyatsoita. The annealing tembiricha yakanyanya. Inobata zvakanyanya kusungwa kwemaprimers kumatemplate uye inoderedza PCR amplification kunyatsoita. Dzimwe nguva zvinodikanwa kushandisa thermometer yakajairika kutarisa denaturation, annealing uye kuwedzera tembiricha muamplifier kana poto inonyungudika nemvura. Ichi zvakare chimwe chezvikonzero zvekutadza kwePCR. Musiyano wekutevedzana kwechinangwa: Kana iyo inoteedzana inoteedzana ikashandurwa kana kudzimwa, izvo zvinokanganisa kusungirirwa kweprimer kune template, kana iyo primer uye template inorasikirwa neanopindirana kutevedzana nekuda kwekudzima kwechimwe chikamu chechinangwa chekutevedzana, PCR amplification. hazvizobudiriri.

2.false positive The PCR amplification bhendi inoratidzika inopindirana nechinangwa chekutevedzana kwebhendi, uye dzimwe nguva bhandi rinonyanya kurongeka uye rakajeka. Zvisina kufanira primer dhizaini: Iyo yakasarudzwa yekumisikidza kutevedzana ine homology neiyo isiri-yakananga amplification kutevedzana, saka kana uchiita PCR amplification, iyo PCR yakasimudzwa chigadzirwa ndeye isiri-yakananga kutevedzana. Kana kutevedzana kwechinangwa kuri kupfupika kana kuti primer ipfupi, manyepo anogona kuitika nyore. Maprimers anofanirwa kuvandudzwa. Muchinjika-kusvibiswa kwekutevedzana kwechinangwa kana zvigadzirwa zvekusimudzira: Pane zvikonzero zviviri zvekusvibiswa uku: Chekutanga, kuchinjika-kusvibiswa kweiyo genome yese kana zvidimbu zvakakura, zvinotungamira kune zvenhema. Iyi yenhema yakanaka inogona kugadziriswa nenzira dzinotevera: Ngwarira uye unyoro paunenge uchishanda kudzivirira kutevedzana kwechinangwa kubva pakuyamwa mupfuti yemuenzaniso kana kupfapfaidzwa kunze kwechubhu yecentrifuge. Kunze kwema enzymes uye zvinhu zvisingakwanise kumisa tembiricha yakakwira, ese mareagents kana zvishandiso zvinofanirwa kucheneswa nekumanikidza kwakanyanya. Machubhu ese ecentrifuge uye matipi ejekiseni ejekiseni anofanira kushandiswa kamwe chete. Kana zvichidikanwa, machubhu ekuita uye mareagents anovhenekerwa nechiedza che ultraviolet asati awedzera chimiro chekuparadza nucleic acids iripo. Chechipiri ndiko kusvibiswa kwezvidimbu zviduku zve nucleic acids mumhepo. Izvi zvimedu zvidiki zvipfupi pane zvakanangwa kutevedzana, asi zvine imwe homology. Zvinogona kupatsanurwa kune mumwe nemumwe, uye mushure mekupindirana kune yekutanga, zvigadzirwa zvePCR zvinogona kukwidziridzwa, zvichikonzera manyepo enhema, ayo anogona kuderedzwa kana kubviswa nedendere PCR nzira.

 

3.Non-specific amplification bands anooneka Mabhendi anoonekwa mushure mePCR amplification haaenderani nehukuru hunotarisirwa, hungave huhombe kana hudiki, kana ese ari maviri mabhendi ekukudza uye asiri chaiwo amplification mabhendi anoonekwa panguva imwe chete. Zvikonzero zvekuonekwa kwemabhandi asiri chaiwo ndezvi: kutanga, maprimers haaenderane zvachose kune yakatarwa kutevedzana, kana maprimers anounganidza kuita dimers. Chikonzero chechipiri ndechekuti iyo Mg2 + ion concentration yakakwira zvakanyanya, tembiricha yekudziya yakadzikira, uye huwandu hwePCR kutenderera hwakanyanya. Chinhu chechipiri kunaka uye kuwanda kweiyo enzyme. Ma Enzymes kubva kune mamwe masosi anowanzo kujaira kune asiri-chaiwo mabhendi asi ma enzymes kubva kune mamwe masosi haadaro. Kuwedzeredza kuwanda kwema enzymes dzimwe nguva kunogona kutungamirira kune kusiri-chaiyo amplification. Iwo ekupikisa anosanganisira: redesign primers kana zvichidikanwa. Deredza huwandu hwe enzyme kana kuitsiva neimwe sosi. Deredza huwandu hwemaprimers, wedzera huwandu hwetemplate nenzira kwayo, uye deredza huwandu hwekutenderera. Wedzera tembiricha yekumisa zvakafanira kana shandisa nzira mbiri-tembiricha (denaturation pa 93°C, annealing uye kuwedzera kutenderera 65°C).

 

4.Flaky drag kana smears inooneka PCR amplification dzimwe nguva inoratidzika semabhendi akasvibiswa, mabhendi akafanana nemashizha kana mabhendi akafanana nekapeti. Zvikonzero zvinowanzo konzerwa ne enzyme yakawandisa kana hurombo husina kunaka hwe enzyme, yakawandisa dNTP concentration, yakawandisa Mg2+ concentration, yakanyanya kuderera annealing tembiricha, uye yakawandisa kutenderera. Matanho ekupikisa anosanganisira: ① Deredza huwandu hwe enzyme, kana kutsiva iyo enzyme neimwe kunobva. ②Deredza kuwanda kwe dNTP. Zvakakodzera kuderedza Mg2 + concentration. Wedzera huwandu hwema templates uye kuderedza huwandu hwema cycles