Siye saba ngumenzi onamava.Ukuphumelela uninzi kwizatifiketi ezibalulekileyo kwimarike yayo kwisaphulelo esikhulu saseTshayina Uvakalelo oluPhezulu lweNqanaba elinye lweProbe Rt-QpcrIKit V2, Umgangatho bubomi befektri, Gxininisa kwimfuno yomthengi ngumthombo wokusinda kunye nophuhliso lwenkampani, Sithobela ukunyaniseka kunye nesimo sengqondo esihle sokusebenza, sijonge phambili ekuzeni kwakho!
Siye saba ngumenzi onamava.Ukuphumelela uninzi kwiziqinisekiso ezibalulekileyo kwimarike yayoChina Taq DNA Polymerase, Qpcr, Inkampani yethu ithembisa: amaxabiso afanelekileyo, ixesha elifutshane lokuvelisa kunye nenkonzo eyanelisayo emva kokuthengisa, siyakwamkela ukuba undwendwele umzi-mveliso wethu nangaliphi na ixesha ofuna.Ndinqwenela ukuba ngoku sineshishini elimnandi nelide kunye !!!

Iinkcazelo

Le khithi isebenzisa inkqubo ye-lysis buffer ekhethekileyo enokuthi ikhulule ngokukhawuleza i-RNA kwiisampulu zeseli ezikhuliswe kwiimpendulo ze-RT-qPCR, ngaloo ndlela iphelisa inkqubo yokucoca i-RNA ethatha ixesha kunye nenzima.Ithemplethi ye-RNA inokufumaneka kwimizuzu nje esi-7.Umxube we-RT oyi-5 × ngokuthe ngqo kunye ne-2 × Direct qPCR Mix-SYBR ii-reagents ezibonelelwe yikhithi zinokufumana ngokukhawuleza nangempumelelo iziphumo zePCR zexesha lokwenyani.

I-5 × Direct RT Mix kunye ne-2 × Direct qPCR Mix-SYBR inokunyamezela inhibitor eqinile, kunye ne-lysate yeesampuli zingasetyenziswa njenge template ye-RT-qPCR ngokuthe ngqo.Le khithi iqulethe i-RNA ephezulu ehambelana ne-Foregene reverse transcriptase, kunye ne-Hot D-Taq DNA polymerase, i-dNTPs, i-MgCl₂, i-reaction buffer, i-PCR optimizer kunye ne-stabilizer.

Iinkcukacha

200×20μl Rxns, 1000×20μl Rxns

Amacandelo ekhithi

Iimpawu&izinto eziluncedo

■ Ilula kwaye isebenzayo : ngeteknoloji ye-Cell Direct RT, iisampuli ze-RNA zinokufumaneka kwimizuzu nje eyi-7.

∎ Imfuno yesampulu incinci, ingaphantsi njengeeseli ezili-10 zinokuvavanywa.

■ Ukugqithisa okuphezulu: inokubona ngokukhawuleza i-RNA kwiiseli ezikhuliswe kwii-384, 96, 24, 12, 6-well plates.

■ I-DNA Eraser inokususa ngokukhawuleza iigenomes ezikhutshiweyo, inciphise kakhulu impembelelo kwiziphumo zovavanyo ezilandelayo.

■ Inkqubo elungiselelwe i-RT kunye ne-qPCR yenza amanyathelo amabini e-RT-PCR akhutshelwe umva asebenze ngakumbi kwaye i-PCR icace ngakumbi, kwaye imelane ngakumbi ne-RT-qPCR reaction inhibitors.

Isicelo seKit

Ububanzi besicelo: iiseli ezikhuliswe.

- I-RNA ekhutshwe ngesampuli ye-lysis: isebenza kuphela kwi-template ye-RT-qPCR yale khithi.

- Ikiti ingasetyenziselwa ezi njongo zilandelayo: uhlalutyo lwe-gene expression, ukuqinisekiswa kwe-siRNA-mediated gene silence effect, ukuhlolwa kweziyobisi, njl.

Umzobo

Ugcino kunye nobomi beShelf

Inxalenye I yale khithi kufuneka igcinwe kwi-4℃;Icandelo II kufuneka ligcinwe kwi -20 ℃.

I-Foregene Protease Plus II kufuneka igcinwe ku-4℃, ungakhenkcezisi ku -20℃.

I-Reagent 2 × Direct qPCR Mix-SYBR kufuneka igcinwe kwi -20 ℃ ebumnyameni;ukuba isetyenziswe rhoqo, inokugcinwa kwi-4 ℃ yokugcina ixesha elifutshane (sebenzisa kwiintsuku ze-10) .Siye saba ngumenzi onamava.Ukuphumelela uninzi lwezatifikethi ezibalulekileyo zentengiso ye-Big isaphulelo se-China High-Sensitivity One-Step Probe Rt-Qpcr Kit V2, Umgangatho bubomi befektri, Gxininisa kwimfuno yomthengi ngumthombo wokusinda kwenkampani kunye nophuhliso, Sithobela ukunyaniseka kunye nesimo sengqondo esihle sokusebenza, sijonge phambili ukuza kwakho!
Isaphulelo esikhuluChina Taq DNA Polymerase, Qpcr, Inkampani yethu ithembisa: amaxabiso afanelekileyo, ixesha elifutshane lokuvelisa kunye nenkonzo eyanelisayo emva kokuthengisa, siyakwamkela ukuba undwendwele i-factory yethu nangaliphi na ixesha olifunayo.Ndinqwenela ukuba ngoku sineshishini elimnandi nelide kunye !!!

QuickEasyTM Cell Direct RT-qI-PCR Kit -Taqman

Ikati.No.DRT-01021/01022

Kwiseli ngqo RT-qPCR usebenzisa ≤ 1000,000 iiseli

Intshayelelo yemveliso

Le mveliso isebenzisa inkqubo ye-lysis buffer ekhethekileyo yokukhulula ngokukhawuleza i-RNA kwiisampuli zeseli ezikhuliswe kwi-RT-qPCR ukuphendula, ukuphelisa ixesha elide kunye nenkqubo yokucoca i-RNA enzima, kunye nemizuzu ye-7 kuphela ukufumana i-template efunekayo ye-RNA, kunye ne-5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman ebonelelwa yikiti ye-quantitive ye-PC ngokukhawuleza

I-5 × Direct RT Mix kunye ne-2 × Direct qPCR Mix-Taqman inonyamezelo oluqinileyo lwe-inhibitor kwaye inokwenza ukuguqulwa okusebenzayo kunye nokukhulisa ngokuthe ngqo usebenzisa i-lysate yesampuli ukuba ilinganiswe njenge template.I-reagent iqulethe i-Foregene Reverse Transcriptase, i-Hot D-Taq DNA Polymerase, i-dNTPs, i-MgCl₂, I-Reaction Buffer, i-PCR Optimizer kunye ne-Stabilizer, enokusetyenziswa kunye ne-lysis buffer ngokukhawuleza kwaye kulula ukubona iisampulu, kwaye ineempawu zobuntununtunu obuphezulu, ukuchaneka kunye nokuzinza.

Iimpawu zemveliso

Itekhnoloji elula, esebenzayo yeCell Direct RT ethatha nje imizuzu esi-7 ukufumana iisampulu zeRNA.

Iimfuno zesampulu zincinci, kwaye ubuncinci beeseli ezili-10 ezikhulisiwe zingasetyenziselwa ukulinga.

Ugqithiso oluphezulu lokufunyanwa kwe-RNA ngokukhawuleza kweeseli ezikhuliswe njenge-384, 96, 24, 12, kunye neepleyiti zequla ezi-6.

I-DNA Eraser iyakwazi ukususa ngokukhawuleza i-genomes ekhutshiweyo, inciphisa kakhulu impembelelo kwiziphumo zovavanyo ezilandelayo.

Iisistim ezilungiselelwe i-RT kunye ne-qPCR zenza i-RT-PCR enamanyathelo amabini aphumelele ngakumbi ukukhuphela okubuyela umva, okucacileyo, kunye ne-RT-qPCR eyomeleleyo ye-reaction inhibitor tolerance.

Isicelo seKit

Ububanzi besicelo: Iiseli ezikhuliswayo.

Isampuli ye-lysis itolikwe i-RNA: isetyenziswe kuphela njenge-template ye-RT-qPCR enamanyathelo amabini.

Iikiti zingasetyenziselwa ezi njongo zilandelayo: uhlalutyo lwe-gene regulatory expression, uvavanyo lwe-allele, ukuhlolwa kweziyobisi, njl.

Ukulinganiselwa kwikhithi

Amaqhekeza anyusiweyo ≤ 300 bp.

Iikiti zisetyenziselwa iiseli ezintsha zenkcubeko.

Ulawulo lomgangatho wemveliso

NgokweNkqubo yoLawulo loMgangatho we- FOREGENE iyonke, ibhetshi nganye yeekhithi ze-Cell Direct RT-qPCR ivavanywa ngokungqongqo amaxesha amaninzi ukuqinisekisa ukuthembeka kunye nokuzinza komgangatho webhetshi nganye yeekhithi.

Imixholo yekhithi

*:I-Cell Lysis, i-5×I-RT Direct Mix, i-2× Direct qPCR Mix-Taqman inokuthengwa ngokwahlukileyo,iinkcukacha zinikezelwe kwiSihlomelo 1 (IPHEPHA 13).

Iimeko zokugcina

1. IiMeko zokuThumela

Yonke inkqubo yothutho lwebhokisi yebhokisi yobushushu obuphantsi, ukuqinisekisa ukuba ikhithi ikwi <4 °C.

2. Iimeko zokugcina

Gcina inxalenye I kwi-4 ° C kunye neCandelo II kwi -20 ° C.

I-Foregene Protease Plus II kufuneka igcinwe kwi-4°C, ingakhenkceziswe ku -20°C.

I-Reagent 2 × Direct qPCR Mix-Taqman igcinwe kwi-20 ° C, okanye kwi-4 ° C yokusetyenziswa kwexesha elifutshane xa isetyenziswe rhoqo (ngaphakathi kweentsuku ze-10).

Ulwazi lwecandelo leKit

I-Buffer CL: Ibonelela ngemekobume efunekayo kwimpendulo yeseli yeseli.

I-Buffer ST: Iyaphelisa into esebenzayo kwi-lysate ukuphepha iziphumo kwi-RT elandelayo.

I-DNA Eraser: I-DNA remover, umphumo wokususa i-genome kwiimvavanyo ezilandelayo.

I-5 × Umxube we-RT ngokuthe ngqo: Iqulethe i-RNA ephezulu ehambelana ne-Foregene Reverse Transcriptase, i-RNase Inhibitor, i-dNTPs, i-stabilizers, iziphuculi, izilungisi, kunye ne-reverse transcription primers ukwenzela ukulungelelaniswa okuphezulu (Random Primer, Oligo (dT)₁₈Isiqalo).

I-Foregene Protease Plus II: Kumxholo we-lysis buffer, iiseli zi-lysed ukukhulula i-nucleic acids.

I-2 × Direct qPCR Mix-Taqman: Le reagent iqulethe iHot D-Taq DNA Polymerase, dNTPs, MgCl₂, i-reaction buffer, i-PCR optimizer, kunye ne-stabilizer.

I-20 × i-ROX Reference Dye: Ngokuqhelekileyo isetyenziswe kwi-Real Time PCR izixhobo zokukhulisa i-ABI, i-Stratagene kunye nezinye iinkampani, isetyenziselwa ukulungelelanisa umahluko phakathi kweetyhubhu ze-PCR kunye neetyhubhu ezibangelwa iimpazamo ze-PCR dosing.I-20 × i-ROX ye-concentration ye-Reference ye-Dye efunekayo kwizixhobo ezahlukeneyo iyahluka, kwaye umsebenzisi unokuyongeza ngokuhambelana noxinzelelo olucetyiswayo lwesixhobo.

RNase-Free ddH₂O: I-RNase-free inzalo yamanzi acocekileyo obuninzi kumanyathelo amabini okusabela kwe-RT-qPCR.

Ukulumkela:(Qinisekisa ukuba ufunda izilumkiso ngononophelo phambi kokuba usebenzise ikhithi)

Nika ingqalelo kwindlela yokusebenza yovavanyo ukuphepha ukungcoliseka phakathi kweesampuli.

Nika ingqalelo ukucoceka kwendawo yovavanyo kunye nezixhobo zokuthintela ukungcoliseka kwe-RNase kunye nokuthotywa kwe-RNA.

Thatha iisampulu zeseli ezitsha okanye ezilondolozwe kakuhle kwaye ungaze usebenzise iisampulu zeseli ezinyibilikisiweyo eziphindaphindiweyo.

I-5 × Direct qPCR Mix, 2× Direct qPCR Mix-Taqman kufuneka igweme ukunyibilika okuphindaphindiweyo, ngaphandle koko kuya kuchaphazela ukukhutshelwa umva kunye nokusebenza kwe-PCR.

Prepaimixheshongaphambiliukusebenza

Qinisekisa ukuba ufunda imiyalelo ngononophelo phambi kokuba usebenzise le khithi.I-Cell Direct RT-qPCR Kit ilula, ifanelekile, kwaye iyakhawuleza ukusebenza, kwaye imiyalelo inika ulwazi olupheleleyo malunga nekhithi yonke kunye nendlela yokuyisebenzisa ngokuchanekileyo.Nceda ulungiselele izixhobo zovavanyo eziyimfuneko kunye nezixhobo ngaphambi kokusetyenziswa.

Izinto zovavanyo kunye nezixhobo

◆ Iiseli zenkcubeko.

◆ I-1.5 ml okanye i-2 ml, i-RNase-/DNase-Free centrifuge tube, i-RNase-/DNase-Free tip, i-0.2 ml sterile qPCR tube.

◆ qPCR machine, pipette, tabletop centrifuge (≥13,400×g) (kuxhomekeke kwiimfuno zovavanyo), njl.

Ukhuseleko

◆ Le mveliso yenzelwe iinjongo zophando lwezenzululwazi kuphela, nceda ungayisebenzisi kwiinjongo zeyeza, iklinikhi, ukutya kunye ne-cosmetic.

◆ Xa usebenzisa imichiza, nxiba iimpahla zaselebhu ezifanelekileyo, iiglavu, iiglasi zokhuselo, njl.

Ukusebenzaizikhokelo

Iinkqubo ze-Cell Lysis, ii-RT systems, kunye ne-qPCR reaction solution supplement packs zingathengwa ngokwahlukeneyo, ngeenkcukacha kwi-Appendix 1 (IPHEPHA 13).

Isikhokelo sokusebenza

A: Ukukhutshwa kwesampulu ye-RNA

I-1.Iiseli zenziwe kwangaphambili: Geza ipleyiti yenkcubeko yeseli nge-PBS ebandayo, emva koko uhlambe iiseli (10-10⁶), 10⁶ kunomlinganiselo weeseli, kuyacetyiswa ukuba Foregene iCell i RNA Isolation Kit iyonke (DE-03111) okanye Animal Total RNA Isolation Kit (DE-03011) for RNA extraction kunye nokucocwa.

1.1.Iiseli ezibambelelayo (24- ipleyiti yequla njengomzekelo)

1.1.1.Qinisekisa inani leeseli kwiqula ngalinye, qaphela ukuba inani leeseli ngu-1 × 10⁵, kwaye usebenzise i-pipette ukususa inkcubeko yenkcubeko kwisitya senkcubeko.

1.1.2.Yongeza i-200 μl ye-1 × ye-PBS epholileyo kwiqula ngalinye.Musa ukubhobhoza ngokuphindaphindiweyo kwaye ususe i-PBS kumaqula.Tilika ipleyiti kwaye ususe iPBS eninzi kangangoko.Qhubekela kwinyathelo lesi-2.

1.1.3.Isitya esahlukileyo senkcubeko yeseli okanye inombolo yereferensi yetafile 1-1 kwisitya senkcubeko yeseli yongezwa i-precooled 1 × PBS yokuhlamba iiseli.

Itheyibhile1-1: Idosi yePBS yamanani ahlukeneyo eeseli

Phawula:Ukuqinisekisa iiseli ezibambelela ngokuqinileyo,inani elikhulu lelahleko yeeseli eziphetshwayo xa uhlamba .

1.2.Iiseli zokunqunyanyiswa okanye iiseli ezibambelelayo ezikhuliswe kwiipleyiti ezingenazo iiporeshi

1.2.1.Iiseli ezibambelelayo ezikhuliswe kwiipleyiti ezingezizo ezininzi (iiseli zokunqunyanyiswa ziqala kwinyathelo elilandelayo 1.2.2), ziqokelele kwaye zahlule iiseli ngokwendlela eqhelekileyo yokuqokelela iiseli, kwaye uzibeke kwipleyiti yenkcubeko okanye ityhubhu ye-centrifuge;ukuba i-trypsinization isetyenzisiwe, ifuna i-centrifugation ukuqokelela iiseli kunye nokususa i-trypsin eseleyo, yongeza iiseli ezibuyiselweyo ze-PBS kwiiseli ezizimeleyo ukuze zisasaze iiseli.

1.2.2.Emva kwenani leeseli ezibaliweyo, iiseli ezicatshulweyo 1 × 10⁵ enye kwiityhubhu ze-centrifuge, ukuqokelela iiseli nge-centrifugation kwi-1000 × g ye-10 min.

1.2.3.Yongeza i-200 μl ye-PBS kwi-tube ye-centrifuge, musa ipayipi ngokuphindaphindiweyo, kwaye unqwenela ngokuthe ngqo i-PBS.qhubela phambili kwisinyathelo sesi-2. (Ukuba kunzima ukukhawuleza kwaye iiseli ziphinde zamiswa kwakhona, zingenziwa i-1000 × g centrifuged 10 min emva kokulahla i-supernatant, i-cell pellet iqhubela phambili kwisinyathelo 2)

I-2.Cell lysis: Susa i-Buffer CL, ubushushu bayo bulinganiswe kwiqondo lokushisa, i-DNA Eraser kunye ne-Foregene Protease Plus II, ngokutsho kwetafile i-1-2 elungiselelwe inkqubo ye-lysis: (Isisombululo se-Lysis silungele ukusetyenziswa).

Itheyibhile 1-2: i-cleavage ukulungiswa kwenkqubo (Qaphela: kulungiselelo lomkhenkce)

3.( 24 –well plate njengomzekelo) I-Pipette 200 μl ye-cell lysis master mix kwiqula ngalinye, Vuthela ngokuphindaphindiweyo izihlandlo ezi-5-10, Fudumeza kwiqondo lobushushu legumbi (20-25 ℃ ) kangangemizuzu emi-5.

Phawula:Ukuze ugweme ukubunjwa kwamaqamza, nceda xa isikali se-pipetting pipette silungelelaniswe kwi-200μl okanye ngaphantsi.Iiseli zingabonakala zinefu emva kwe-lysis, into eqhelekileyo.

4.(i-24-well plate njengomzekelo) yongezwa kwi-liquid 20 μl Buffer ST (iinkqubo ezahlukeneyo ze-lysis Buffer ST eyongeziweyo kwisixa esiboniswe kwiThebhile 1-3), i-pipetting ephindaphindiweyo ngamaxesha angama-5-10, kwiqondo lokushisa kwegumbi (20-25 ℃) yafukanyelwa i-2 min.

Phawula:Ingqungquthela yepipette ichithwa ngaphantsi kwendawo, iqinisekisa ukuba i-lysate yongezwa,ukuphepha ukubunjwa kwamaqamza, nceda xa isikali sepipetting pipette sihlengahlengiswe sibe yi-200μl okanye ngaphantsi.

Uluhlu 1-3:Yongeza i-Buffer ST

5.I-lysate isetyenziselwa imifuniselo ye-RT-qPCR elandelayo.Ukuba imifuniselo elandelayo ayinakwenziwa ngexesha, nceda uyigcine emkhenkceni ingabi ngaphezu kwe-2hr, kwaye uyigcine ku -20 ℃ okanye -80 ℃ (kungekho ngaphezu kweenyanga ezintathu).

B: Ukulungiswa kwenkqubo ye-RT

1.Thatha i-5 × Direct RT Mix kwaye uyibeke kwindawo yokuhlamba i-ice, yiyeke inyibilike ngokwemvelo, kwaye uyixube ngobumnene ukuze uyisebenzise kamva;khupha i-ddH2O yamahala yeRNase kwaye uyinyibilikise kwaye uyibeke kwindawo yokuhlambela yomkhenkce ukuze isetyenziswe kamva.Lungiselela inkqubo yokusabela kumkhenkce ngokweTheyibhile 2-1 engezantsi.

Itheyibhile 2-1: Ukulungiswa kwenkqubo yokusabela kwe-RT

2.Emva kokugqitywa kokwenziwa kwenkqubo, ixutywe ngobumnene kunye ne-centrifuged ngokufutshane kwitheyibhile ilandelayo 2 -2 iimeko zokuphendula i-RT reaction.

Itheyibhile 2-2: Ukumiswa kwemeko ye-Reaction ye-RT

3.Emva kokugqitywa kwempendulo, imveliso yokusabela ibekwe ngqo kwi-ice qPCR, nceda ubeke ukugcinwa kwexesha elide -20 ℃ okanye -80 ℃.

Qaphela: Ngenxa yokusetyenziswa kwetemplate engacocwanga, i-white precipitates inokuvela kwi-reverse transcription product.Le yinto eqhelekileyo.I-Centrifuge i-supernatant ngokukhawuleza kwiimvavanyo ezilandelayo.

Isiphumo Isisombululo reaction RT yongezwa kwinyathelo elilandelayo Iinkqubo yokusabela PCR Ixesha lokwenyani, kuyacetyiswa ukuba ukongeza iimali ukusuka 10-30% inkqubo reaction.

C: Ukulungiswa kwenkqubo yokusabela kwe-qPCR

1.Isixa esifanelekileyo se-B esilungiselelwe kwithemplethi ye-cDNA yenyathelo ngokwale tafile ilandelayo 3-1 ukulungiselela inkqubo yokusabela.

Qaphela: Ubungakanani be-cDNA template akhawunti kwi-10-30% yenkqubo ye-qPCR.Umzekelo, kwi-20μl qPCR inkqubo, yongeza i-2-6 μl ye-lysis buffer, kodwa ingabi ngaphezu kwe-6 μl.

2.The optimization iimeko ezilungileyo qPCR (Annealing ubushushu, njalo njalo) ukwenzela qPCR reaction (Iimeko zokusabela ezinikwe kwiThebhile 3-2).

Qaphela: Zama ukusebenzisa iimeko ezilungiselelwe ukusabela kwe-qPCR ukufumana iziphumo ezingcono.

Itheyibhile 3-1: Ukulungiswa kwenkqubo yokusabela kwe-PCR

I-1 *: Ugxininiso lwe-Primer lunokulungelelaniswa kuluhlu lwe-50-900 nM xa ukusebenza kwe-primer reaction kungalunganga.

Qaphela: Inkqubo ye-qPCR inokulungelelaniswa ngokweemfuno zovavanyo kunye nemodeli yebhayisikile ye-fluorescence.Kwi-qPCR kwi-50μl inkqubo, lungisa umthamo we-reagent ngokulinganayo ngokwe-20μl inkqubo.

I-2*: Khetha i-concentration yokugqibela efanelekileyo ye-ROX Reference Dye ngokwe-fluorescence quantitative thermal cycler.Olona gxininiso lwe-ROX Reference Dye lwe-fluorescence quantitative cyclers luboniswe kwitheyibhile engezantsi:

Itheyibhile 3-2: Iimeko zokusabela kwe-qPCR zinikezelwe

Qaphela: Ukuze ufumane esona siphumo se-qPCR, igradient PCR ingasetyenziselwa ukunyusa iimeko zokusabela kwiitemplates ezahlukeneyo kunye neeprimers ezahlukeneyo.Iimeko zokusabela kwe-PCR ziyahluka ngokuxhomekeke kumhlalutyi we-fluorescence, itemplate, i-primer, njl. Kwintsebenzo ethile, iimeko zokusabela ezifanelekileyo kufuneka ziyilwe ngokwemiqathango ethile ye-fluorescence quantitative thermal cycler, uhlobo lwetemplate, ubungakanani beqhekeza lomdla, ukulandelelana kwesiseko seqhekeza elikhutshiweyo kunye nobude bexesha, kunye nobude be-GC, kunye nobude bexesha lobushushu, kunye nobude bexesha lobushushu, kunye nobude be-GC.

Ixesha lokwenyani imigaqo yoyilo lwe-PCR primer

I-Primer yokuPhambili kunye ne-Primer yokuBuyisa

Ngexesha lokwenyani iPCR, uyilo lweprimer lubaluleke kakhulu.Iiprimers zinxulumene nobucacileyo kunye nokusebenza kakuhle kokwandiswa kwePCR, kwaye zinokuyilwa ngokubhekiselele kule migaqo ilandelayo:

◆ Ubude be-Primer: 18-30bp.

◆ Umxholo we-GC: 40-60%.

◆ Ixabiso le-Tm: I-software yoyilo lwe-Primer, njenge-Primer 5, inokunika ixabiso le-Tm le-primer.Amaxabiso e-Tm ee-primers ezinyukayo nezisezantsi kufuneka zibe kufutshane kangangoko.Ifomula yokubala ye-Tm nayo ingasetyenziswa: Tm = 4 °C (G + C) + 2 °C (A + T).Xa kusenziwa i-PCR, iqondo lobushushu elingaphantsi kwexabiso le-primer Tm le-5 °C likhethwa ngokubanzi njengeqondo lobushushu lokutshisa (ukunyuka okuhambelanayo kwiqondo lobushushu lobushushu kunokunyusa ukuchana kwe-PCR reaction).

◆ Iiprimers kunye neemveliso zePCR:

◆ I-design primer PCR yokukhulisa ubude bemveliso ikhethwa yi-100-150bp.

◆ Iiprayimari zoyilo kwindawo yesibini yesakhiwo sethemplethi kufuneka ziphetshwe kangangoko kunokwenzeka.

◆ Gwema ukuqulunqwa kwe-2 okanye iziseko ezingaphezulu ezihambelanayo phakathi kwee-3 'end of the upstream and downstream primers.

◆ Isiseko se-Primer 3′ asikwazi ukubakho kunye ne-3 eyongezelelweyo elandelelanayo ye-G okanye i-C.

◆ Iiprimers ngokwazo azikwazi ukuba nezakhiwo ezincedisayo, ngaphandle koko isakhiwo se-hairpin siya kwenziwa, esichaphazela ukukhulisa i-PCR.

◆ I-ATCG kufuneka ihanjiswe ngokulinganayo ngokulandelelana kwe-primer, kwaye isiseko se-terminal ye-3 kufuneka sigwenywe njenge-T.

Isihlomelo1:Cell ngqoI-RT-qPCR Ikhithi icandeloIpakethi yokongeza

1.Cell Lysis Solution