Sibe nomkhiqizi onolwazi.Ukuzuza iningi ekutholeni izitifiketi ezibalulekile zemakethe yayo ye-Big Sensitivity China High Sensitivity One-Step Probe Rt-QpcrI-Kit V2, Ikhwalithi iyimpilo yefekthri, Gxila ekufuneni kwamakhasimende kuwumthombo wokusinda nentuthuko yenkampani, Sinamathela ekuthembekeni nasekusebenzeni okuhle ngokholo, sibheke phambili ekufikeni kwakho!
Sibe nomkhiqizi onolwazi.Ukuzuza iningi ezitifiketini ezibalulekile zemakethe yayoI-China Taq DNA Polymerase, Qpcr, Inkampani yethu ithembisa: amanani afanele, isikhathi esifushane sokukhiqiza kanye nenkonzo eyanelisayo yangemva kokuthengisa, siyakwamukela futhi ukuthi uvakashele imboni yethu nganoma yisiphi isikhathi osifunayo.Ngifisa manje sinebhizinisi elimnandi nelide ndawonye !!!

Izincazelo

Le khithi isebenzisa isistimu ye-lysis buffer eyingqayizivele engakhulula ngokushesha i-RNA kumasampula eseli akhulisiwe ukuze kusabela i-RT-qPCR, ngaleyo ndlela iqede inqubo yokuhlanza i-RNA edla isikhathi nekhandlayo.Isifanekiso se-RNA singatholakala ngemizuzu engu-7 nje.I-5×Direct RT Mix kanye ne-2×Direct qPCR Mix-SYBR ama-reagents anikezwe ikhithi angathola ngokushesha nangempumelelo imiphumela ye-PCR yobuningi besikhathi sangempela.

I-5×Direct RT Mix kanye ne-2×Direct qPCR Mix-SYBR inokubekezelela okuqinile kwe-inhibitor, futhi i-lysate yamasampuli ingasetshenziswa njengesifanekiso se-RT-qPCR ngokuqondile.Le khithi iqukethe i-RNA high-affinity Foregene reverse transcriptase, kanye ne-Hot D-Taq DNA polymerase, dNTPs, MgCl₂, isigcinalwazi sokusabela, i-PCR optimizer nesiqinisi.

Imininingwane

200×20μl Rxns, 1000×20μl Rxns

Izingxenye zekhithi

Izici&izinzuzo

■ Kulula futhi kusebenza ngempumelelo : Ngobuchwepheshe be-Cell Direct RT, amasampula e-RNA angatholwa ngemizuzu engu-7 nje.

■ Isidingo sesampula sincane, siphansi njengoba amaseli ayi-10 angahlolwa.

■ Ukuphuma okuphezulu: ikwazi ukubona ngokushesha i-RNA kumaseli akhiwe kumapuleti angu-384, 96, 24, 12, 6-wemithombo.

■ I-DNA Eraser ingasusa ngokushesha ama-genome akhululiwe, inciphise kakhulu umthelela emiphumeleni yokuhlola elandelayo.

■ Isistimu ye-RT ethuthukisiwe kanye ne-qPCR yenza ukuloba okuhlanekezela okuzinyathelo ezimbili kwe-RT-PCR kusebenze kahle futhi i-PCR icace kakhulu, futhi imelane kakhulu ne-RT-qPCR reaction inhibitors.

Isicelo sekhithi

Ububanzi bokusebenza: amaseli akhulisiwe.

- I-RNA ekhishwe ngesampula ye-lysis: isebenza kuphela kusifanekiso se-RT-qPCR sale kit.

- Ikhithi ingasetshenziselwa lezi zinhloso ezilandelayo: ukuhlaziywa kwesisho sofuzo, ukuqinisekiswa komphumela wokuthulisa ufuzo we-siRNA-mediated, ukuhlolwa kwezidakamizwa, njll.

Umdwebo

Isitoreji kanye Nempilo yeshelufu

Ingxenye I yale kit kufanele igcinwe ku-4℃;Ingxenye II kufanele igcinwe ku -20 ℃.

I-Foregene Protease Plus II kufanele igcinwe ku-4℃, ingafrizi ku -20℃.

I-Reagent 2×Direct qPCR Mix-SYBR kufanele igcinwe ku -20℃ ebumnyameni;uma isetshenziswa njalo, ingabuye igcinwe ku-4℃ ukuze igcinwe isikhathi esifushane (isetshenziswe phakathi kwezinsuku eziyi-10).Sibe nomkhiqizi onolwazi.Ukuzuza iningi ekutholeni izitifiketi ezibalulekile zemakethe yayo yesaphulelo Esikhulu I-China High Sensitivity One-Step Probe Rt-Qpcr Kit V2, Ikhwalithi iyimpilo yefekthri, Gxila ekufuneni kwamakhasimende kuwumthombo wokusinda nokuthuthuka kwenkampani, Sinamathela ekuthembekeni nasekusebenzeni okuhle ngokholo, sibheke phambili ekufikeni kwakho!
Isaphulelo esikhuluI-China Taq DNA Polymerase, Qpcr, Inkampani yethu ithembisa: amanani afanele, isikhathi esifushane sokukhiqiza kanye nenkonzo eyanelisayo yangemva kokuthengisa, siyakwamukela futhi ukuthi uvakashele imboni yethu nganoma yisiphi isikhathi ofuna ngaso.Ngifisa manje sinebhizinisi elimnandi nelide ndawonye !!!

QuickEasyTM Cell Direct RT-qI-PCR Kit -Taqman

I-Cat.No.DRT-01021/01022

Kuseli eliqondile i-RT-qPCR lisebenzisa amaseli angu-≤ 1000,000

Isingeniso somkhiqizo

Lo mkhiqizo usebenzisa isistimu ye-lysis buffer eyingqayizivele ukuze ukhulule ngokushesha i-RNA kumasampula eseli akhulisiwe ukuze kusabela i-RT-qPCR, uqede inqubo yokuhlanza i-RNA ethatha isikhathi futhi ekhandlayo, kanye nemizuzu engu-7 kuphela ukuthola ithempulethi edingekayo ye-RNA, nge-5× Direct RT Mix, 2× Direct qPCR Mix-Taqman thola imiphumela ye-qualyntitive PCR ngokushesha nangendlela efanele.

I-5× Direct RT Mix kanye ne-2× Direct qPCR Mix-Taqman zinokubekezelela okuqinile kwe-inhibitor futhi zingenza ukuguqulwa okuphumelelayo nokukhulisa ukukhulisa okuthile kusetshenziswa i-lysate yesampula ukuze ikalwe njengesifanekiso.I-reagent iqukethe i-Foregene Reverse Transcriptase, i-Hot D-Taq DNA Polymerase, i-dNTPs, i-MgCl₂, I-Reaction Buffer, i-PCR Optimizer ne-Stabilizer, engasetshenziswa ne-lysis buffer ukuthola ngokushesha futhi kalula amasampula, futhi inezici zokuzwela okuphezulu, ukucaciswa nokuzinza.

Izici zomkhiqizo

Ubuchwepheshe obulula, obusebenzayo be-Cell Direct RT obuthatha imizuzu embalwa nje eyi-7 ukuthola amasampula e-RNA.

Izidingo zesampula zincane, futhi ubuncane bamaseli akhulisiwe angu-10 angasetshenziselwa ukuhlolwa.

Ukuphuma okuphezulu kokutholwa ngokushesha kwe-RNA kwamaseli akhulisiwe njengamapuleti angu-384, 96, 24, 12, kanye namapuleti emithombo engu-6.

I-DNA Eraser iyakwazi ukususa ngokushesha ama-genome akhululiwe, inciphisa kakhulu umthelela emiphumeleni yokuhlola elandelayo.

Amasistimu e-RT athuthukisiwe kanye ne-qPCR anika amandla i-RT-PCR enezinyathelo ezimbili ngokubhala okubuyela emuva okusebenza kahle kakhudlwana, ukucaciswa, nokubekezelela okuqinile kwe-RT-qPCR ukusabela kwe-inhibitor.

Isicelo sekhithi

Ububanzi bokusebenza: Amaseli akhiwe.

Isampula ye-lysis ehunyushwe i-RNA: isetshenziswa kuphela njengezinyathelo ezimbili zesifanekiso se-RT-qPCR.

Amakhithi angasetshenziselwa lezi zinhloso ezilandelayo: ukuhlaziywa kwenkulumo yokulawula izakhi zofuzo, ukuhlolwa kwe-allele, ukuhlolwa kwezidakamizwa, njll.

Ukulinganiselwa kwekhithi

Izingcezu ezikhulisiwe ≤ 300 bp.

Amakhithi asetshenziselwa amaseli amasiko amasha.

Ukulawulwa kwekhwalithi yomkhiqizo

Ngokuvumelana ne-ForEGENE's Total Quality Management System, iqoqo ngalinye lamakhithi ochungechunge lwe-Cell Direct RT-qPCR ihlolwa ngokuqinile izikhathi eziningi ukuze kuqinisekiswe ukwethembeka nokuzinza kwekhwalithi yeqoqo ngalinye lamakhithi.

Okuqukethwe kwekhithi

*:I-Cell Lysis, 5×Direct RT Mix, 2× Direct qPCR Mix-Taqman angathengwa ngokuhlukana, imininingwane inikezwe kuSithasiselo 1 (IKHASI 13).

Izimo zokugcina

1. Izimo Zokuthumela

Yonke inqubo yokuthutha kwebhokisi leqhwa lezinga lokushisa eliphansi, ukuqinisekisa ukuthi ikhithi isesimweni esingu-<4 °C.

2. Izimo zokugcina

Gcina ingxenye I ku-4°C kanye neNgxenye II ku- -20°C.

I-Foregene Protease Plus II kufanele igcinwe ku-4°C, hhayi iqhwa ku -20°C.

I-Reagent 2× Direct qPCR Mix-Taqman igcinwa ku -20°C, noma ku-4°C ukuze isetshenziswe isikhathi esifushane uma isetshenziswa njalo (zingakapheli izinsuku eziyi-10).

Ulwazi lwengxenye yekhithi

I-Buffer CL: Ihlinzeka ngendawo edingekayo yokusabela kwe-cell lysis.

I-Buffer ST: Inqamula into esebenzayo ku-lysate ukugwema imiphumela ku-RT elandelayo.

I-DNA Eraser: Isikhiphi se-DNA, umphumela wokukhipha i-genome ekuhloleni okwalandela.

I-5× Direct RT Mix: Iqukethe ukuhlobana okuphezulu kwe-RNA i-Foregene Reverse Transcriptase, i-RNase Inhibitor, i-dNTPs, iziqinisi, izithuthukisi, izilungiseleli, neziqalisi zokuhlehla zokulotshwa ukuze kuhambisane kahle (Random Primer, Oligo(dT)₁₈Isiqalo).

I-Foregene Protease Plus II: Kumongo we-lysis buffer, amaseli ayahluzwa ukuze akhulule ama-nucleic acid.

I-2× Direct qPCR Mix-Taqman: Lesi sikhungo siqukethe i-Hot D-Taq DNA Polymerase, i-dNTPs, i-MgCl₂, isigcinalwazi sokusabela, i-PCR optimizer, nesiqinisi.

I-20 × ROX Reference Dye: Ngokuvamile isetshenziswa kumathuluzi wokukhulisa we-Real Time PCR we-ABI, Stratagene nezinye izinkampani, isetshenziselwa ukulungisa umehluko phakathi kwamashubhu e-PCR namashubhu abangelwa amaphutha wokudosa kwe-PCR.Ukugxiliswa kwe-20 × ROX Reference Dye okudingekayo kumathuluzi ahlukene kuhlukile, futhi umsebenzisi angakwazi ukuyengeza ngokuya ngokugxila okunconyiwe kwethuluzi.

I-ddH yamahhala ye-RNase₂O: Amanzi ahlanzekile ahlanzekile angenayo i-RNase ezinyathelo ezimbili zokusabela kwe-RT-qPCR.

Izinyathelo zokuzivikela:(Qinisekisa ukuthi ufunda izinyathelo zokuphepha ngokucophelela ngaphambi kokusebenzisa ikhithi)

Naka indlela yokusebenza yokuhlolwa ukuze ugweme ukungcoliswa phakathi kwamasampuli.

Naka ukuhlanzeka kwendawo yokuhlola kanye nezitsha ukuze ugweme ukungcoliswa kwe-RNase kanye nokonakaliswa kwe-RNA.

Thatha amasampula eseli amasha noma alondolozwe kahle futhi ungalokothi usebenzise amasampula eseli aqandisiwe ancibilikisiwe.

I-5× Direct qPCR Mix,2× Direct qPCR Mix-Taqman kufanele igweme ukuncibilika kweqhwa okuphindaphindiwe, ngaphandle kwalokho kuzophazamisa ukuloba okuhlanekezelwe nokusebenza kahle kwe-PCR.

Prepaizabelongaphambiliukusebenza

Qiniseka ukuthi ufunda imiyalo ngokucophelela ngaphambi kokusebenzisa le kit.I-Cell Direct RT-qPCR Kit ilula, ilula, futhi iyashesha ukusebenza, futhi imiyalelo inikeza ulwazi oluphelele mayelana nayo yonke ikhithi nokuthi isetshenziswa kanjani ngendlela efanele.Sicela ulungise izinto zokuhlola ezidingekayo kanye namathuluzi ngaphambi kokusetshenziswa.

Izinto zokuhlola nezinto zokusebenza

◆ Amaseli Amasiko.

◆ 1.5 ml noma 2 ml, RNase-/DNase-Free centrifuge tube, RNase-/DNase-Free ithiphu, 0.2 ml oyinyumba qPCR tube.

◆ qPCR umshini, pipette, tabletop centrifuge (≥13,400×g) (kuye ngezidingo zokuhlola), njll.

Ukuphepha

◆ Lo mkhiqizo ngowezinjongo zocwaningo lwesayensi kuphela, sicela ungawusebenziseli izinjongo zemithi, ezomtholampilo, zokudla nezimonyo.

◆ Lapho usebenzisa amakhemikhali, gqoka izingubo zaselabhu ezifanele, amagilavu, izibuko zokuzivikela, njll.

Ukusebenzaabaqondisi

Amasistimu e-Cell Lysis, amasistimu e-RT, namaphakethe esixazululo se-qPCR angathengwa ngokuhlukana, ukuze uthole imininingwane kuSithasiselo 1 (IKHASI 13).

Umhlahlandlela wokusebenza

A: Isampula yokukhishwa kwe-RNA

1.Amaseli enziwe ngaphambili: Geza ipuleti le-cell culture nge-PBS ebandayo, bese uhlanza amaseli (10-10⁶), 10⁶ kunenani lamaseli, kuyanconywa Foregene the Cell an RNA Isolation Kit the Total (DE-03111) noma Animal Total RNA Isolation Kit (DE-03011) ukuze kukhishwe i-RNA nokuhlanzwa.

1.1.Amaseli anamathelayo (ipuleti lemithombo engama-24 njengesibonelo)

1.1.1.Nquma inani lamaseli emthonjeni ngamunye, thola ukuthi inani lamaseli ngu-1 × 10⁵, futhi usebenzise i-pipette ukuze ususe isiko lamasiko esitsheni samasiko.

1.1.2.Engeza u-200 μl we-1 × PBS epholile ngaphambili emthonjeni ngamunye.Musa ukubhobhoza ngokuphindaphindiwe futhi ususe i-PBS emithonjeni.Tshekisa ipuleti futhi ususe i-PBS eningi ngangokunokwenzeka.Qhubekela esinyathelweni sesi-2.

1.1.3.Isitsha esihlukile se-cell culture noma ithebula lenombolo yereferensi 1-1 endishini ye-cell culture yengezwe i-1 × PBS yokuwashwa kwamaseli ngaphambili.

Ithebula1-1: Umthamo we-PBS wezinombolo ezihlukene zamaseli

Qaphela:Ukuqinisekisa amaseli anamathelayo aqinile,inani elikhulu lokulahlekelwa kwamaseli kugwenywe lapho ugeza .

1.2.Amaseli amisiwe noma amaseli anamathelayo akhuliswe kumapuleti angenazo izimbotshana

1.2.1.Amaseli anamathelayo akhuliswe kumapuleti angewona ama-multi-chamber (amangqamuzana amisiwe aqala esinyathelweni esilandelayo 1.2.2), aqoqe futhi ahlukanise amaseli ngendlela evamile yokuqoqa amaseli, futhi awabeke epuletini lesiko noma ishubhu le-centrifuge;uma i-trypsinization isetshenziswa, idinga i-centrifugation ukuze iqoqe amaseli futhi ikhiphe i-trypsin esele, yengeza amaseli amiswe kabusha e-PBS kumaseli ngamanye ukuze ahlakaze amaseli.

1.2.2.Ngemuva kwenani lamaseli abaliwe, amaseli acashuniwe 1×10⁵ eyodwa kuya kumashubhu e-centrifuge, ukuqoqa amaseli nge-centrifugation ku-1000 × g imizuzu engu-10.

1.2.3.Engeza u-200 μl PBS kushubhu ye-centrifuge, ungabhobhozi ngokuphindaphindiwe, futhi ulangazelele i-PBS ngokuqondile.qhubekela esinyathelweni sesi-2. (Uma kunzima ukwehla futhi amaseli aphinde amiswa futhi, angenziwa 1000×g centrifuged 10 min ngemva kokulahla amandla amakhulu, i-cell pellet iqhubekela esinyathelweni sesi-2)

I-2.Cell lysis: Khipha i-Buffer CL, izinga lokushisa layo elilingana negumbi lokushisa, i-DNA Eraser kanye ne-Foregene Protease Plus II, ngokusho kwethebula elilandelayo i-1-2 elungiselelwe uhlelo lwe-lysis: (Isixazululo se-Lysis sesilungele ukusetshenziswa).

Ithebula 1-2: i-cleavage ukulungiswa kwesistimu (Qaphela: ekulungiseni eqhweni)

3.( 24 –ipuleti lomthombo njengesibonelo) I-Pipette 200 μl ye-cell lysis master mix emthonjeni ngamunye, Vuthela ngokuphindaphindiwe izikhathi ezingu-5-10, Fudumeza endaweni yokushisa (20-25 ℃) imizuzu emi-5.

Qaphela:Ukuze ugweme ukwakheka kwamabhamuza, sicela lapho isikali se-pipette silungiswa sibe ngu-200μl noma ngaphansi.Amaseli angase abonakale eguqubele ngemva kwe-lysis, okuyinto evamile.

4.(24 –well plate njengesibonelo) yengezwe oketshezini 20 μl I-Buffer ST (izinhlelo ze-lysis ezihlukene I-Buffer ST engezwe ngenani eliboniswe kuThebula 1-3), ukubhoboza kwamapayipi okuphindaphindiwe izikhathi ezingu-5-10, ekamelweni lokushisa (20-25 ℃) zafukanyelwa imizuzu emi-2.

Qaphela:Ithiphu ye-pipette ilahlwa ngaphansi kwendawo, iqinisekisa ukuthi i-lysate yanezelwa,ukugwema ukwakheka kwamabhamuza, sicela lapho isikali se-pipette silungiswa sibe ngu-200μl noma ngaphansi.

Ithebula 1-3:Engeza ibhafa ST

5.I-lysate isetshenziselwa ukuhlolwa okulandelayo kwe-RT-qPCR.Uma ukuhlolwa okulandelayo kungeke kwenziwe ngesikhathi, sicela ukugcine eqhweni isikhathi esingeqile ku-2hr, futhi ukugcine ku -20℃ noma -80 ℃ (zingabi ngaphezu kwezinyanga ezintathu).

B: Ukulungiswa kohlelo lwe-RT

1.Khipha i-5 × Direct RT Mix bese uyibeka endaweni yokugeza yeqhwa, yiyeke inyibilike ngokwemvelo, bese uyixuba ngobumnene ukuze isetshenziswe kamuva;khipha i-ddH2O yama-RNase-Free bese uyincibilikisa bese uyibeka endaweni yokugeza eqhweni ukuze isetshenziswe kamuva.Lungiselela uhlelo lokusabela eqhweni ngokweThebula 2-1 ngezansi.

Ithebula 2-1: Ukulungiswa kwesistimu yokusabela ye-RT

2.Ngemva kokuqedwa kokwakhiwa kwesistimu, kuxutshwe ngobumnene futhi kufakwe i-centrifuged kafushane kuthebula elilandelayo 2 -2 izimo zokusabela ukusabela kwe-RT.

Ithebula 2-2: Ukulungiselelwa kwesimo sokusabela kwe-RT

3.Ngemva kokuqedwa kokusabela, umkhiqizo wokusabela ubekwe ngqo eqhweni le-qPCR, sicela ubeke ukulondolozwa kwesikhathi eside -20℃ noma -80 ℃.

Qaphela: Ngenxa yokusetshenziswa kwesifanekiso esingahlanjululwanga, imvula emhlophe ingase ivele kumkhiqizo wokuloba okuhlanekezelwe.Lokhu kuyinto evamile.I-Centrifuge i-supernatant ngokushesha ukuze uthole izivivinyo ezilandelayo.

Isixazululo sokusabela se-RT esiwumphumela sengezwe ezinhlelweni zokusabela ze-PCR ze-Isinyathelo Sangempela Sesikhathi Sangempela, kunconywa ukuthi wengeze amanani asukela ku-10-30% wesistimu yokusabela.

C: ukulungiswa kwesistimu yokusabela kwe-qPCR

1.Inani elifanele lika-B elilungiswe esifanekiso se-cDNA sesinyathelo ngokusho kwethebula elilandelayo 3-1 ukuze kulungiswe uhlelo lokusabela.

Qaphela: Inani lesifanekiso se-cDNA libalelwa ku-10-30% wohlelo lwe-qPCR.Isibonelo, kusistimu ye-qPCR engu-20μl, engeza u-2-6 μl webhafa ye-lysis, kodwa ungadluli ku-6 μl.

2.Izimo ezinhle ze-qPCR (ukushisa kwe-Annealing, njll.) zokusabela kwe-qPCR (Izimo zokusabela ezinikezwe kuThebula 3-2).

Qaphela: Zama ukusebenzisa izimo ezilungiselelwe ukusabela kwe-qPCR ukuze uthole imiphumela engcono.

Ithebula 3-1: Ukulungiswa kwesistimu yokusabela ye-PCR

I-1*: Ukugxiliswa kwe-Primer kungalungiswa kububanzi obungu-50-900 nM lapho ukusebenza kokusabela kwe-primer kungalungile.

Qaphela: Uhlelo lwe-qPCR lungalungiswa ngokuya ngezidingo zokuhlola kanye nemodeli yomjikelezo we-fluorescence.Kwe-qPCR ku-50μl, lungisa umthamo we-reagent ngokulingana ngokuya kwe-20μl uhlelo.

2*: Khetha ukugxiliswa kokugcina okufanele kwe-ROX Reference Dye ngokuya nge-fluorescence quantitative thermal cycler.Ukugxila okufanele kakhulu kwe-ROX Reference Dye kwamabhayisikili omthamo we-fluorescence ajwayelekile kukhonjisiwe kuthebula elingezansi:

Ithebula 3-2: Izimo zokusabela ze-qPCR zinikeziwe

Qaphela: Ukuze uthole umphumela ongcono kakhulu we-qPCR, i-gradient PCR ingasetshenziswa ukuthuthukisa izimo zokusabela zezifanekiso ezihlukene neziqalo ezihlukene.Izimo zokusabela ze-PCR ziyahlukahluka kuye ngesihlazi se-fluorescence, isifanekiso, i-primer, njll. Emsebenzini othize, izimo zokusabela ezifanele zidinga ukuklanywa ngokuya ngezimo ezithile ze-fluorescence quantitative thermal cycler, uhlobo lwesifanekiso, usayizi wocezu lwenzuzo, ukulandelana kwesisekelo kwesiqephu se-amplified ye-amplified ye-GC, ubude besikhathi, njll.

Izimiso ze-Real Time PCR primer design

Dlulisela phambili i-Primer kanye ne-Primer Reverse

Nge-Real Time PCR, ukuklama kokuqala kubaluleke kakhulu.Ama-Primers ahlobene nokucaciswa nokusebenza kahle kokukhulisa i-PCR, futhi angaklanywa ngokubhekiselwa kule migomo elandelayo:

◆ Ubude be-Primer: 18-30bp.

◆ Okuqukethwe kwe-GC: 40-60%.

◆ Inani le-Tm: Isofthiwe yokuklama i-Primer, njenge-Primer 5, inganikeza inani le-Tm le-primer.Amanani e-Tm okuqalisa akhuphuka nomfula kufanele abe seduze ngangokunokwenzeka.Ifomula yokubala ye-Tm nayo ingasetshenziswa: Tm = 4 °C (G + C) + 2 °C (A + T).Lapho kwenziwa i-PCR, izinga lokushisa elingaphansi kwenani lokuqala le-Tm elingu-5 °C livame ukukhethwa njengezinga lokushisa lokukhipha isisu (ukwenyuka okuhambisanayo kwezinga lokushisa le-anneal kungakhuphula ukucaciswa kokusabela kwe-PCR).

◆ Imikhiqizo ye-Primers ne-PCR:

◆ I-design primer PCR ubude bomkhiqizo wokukhulisa u-100-150bp.

◆ Iziqalo zokuklama endaweni yesibili yesakhiwo sesifanekiso kufanele zigwenywe ngangokunokwenzeka.

◆ Gwema ukwakheka kwezisekelo ezi-2 noma ngaphezulu ezihambisanayo phakathi kwemikhawulo engu-3′ yeziqalisi ezikhuphuka nomfula.

◆ Itheminali ye-Primer 3′ ayikwazi ukuba khona nezinye ezingu-3 ezengeziwe ezilandelanayo ze-G noma u-C.

◆ Ama-primer ngokwawo awakwazi ukuba nezakhiwo ezihambisanayo, ngaphandle kwalokho kuzokwakhiwa isakhiwo se-hairpin, esithinta ukukhuliswa kwe-PCR.

◆ I-ATCG kufanele isatshalaliswe ngokulinganayo ngangokunokwenzeka ekulandeleni kokuqala, futhi isisekelo setheminali esingu-3′ kufanele sigwenywe njengo-T.

Isithasiselo1:Cethi DirectI-RT-qPCR Ingxenye yekhithit iphakethe le-supplement

1.Cell Lysis Solution