Sixhomekeke kumandla obugcisa owomeleleyo kwaye sihlala sisenza itekhnoloji ephucukileyo ukwanelisa imfuno ye-OEM China Rt-PCR Mix yeQpcr, Intsebenziswano enyanisekileyo kunye nawe, lilonke liya kuphuhla ngolonwabo ngomso!
Sixhomekeke kumandla obugcisa owomeleleyo kwaye sihlala sisenza itekhnoloji ephucukileyo ukwanelisa iimfunoChina Taq DNA Polymerase, Qpcr, Ngoku, sinikezela ngobuchwephesha abathengi kunye nezisombululo zethu eziphambili Kwaye ishishini lethu alikona nje kuphela "ukuthenga" kunye "nokuthengisa", kodwa kugxininise ngakumbi.Sijonge ukuba ngababoneleli bakho abathembekileyo kunye nentsebenziswano yexesha elide eTshayina.Ngoku, sinethemba lokuba singabahlobo nawe.

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 isetyenziswa rhoqo, inokugcinwa kwi-4℃ yokugcina ixesha elifutshane (usebenzise kwiintsuku ezili-10) sixhomekeke kumandla obugcisa aqinileyo kwaye ngokuqhubekayo sidala ubugcisa obuntsonkothileyo ukwanelisa imfuno ye-OEM China Rt-PCR Mix forQpcr, Intsebenziswano enyanisekileyo kunye nawe, lilonke liya kuphuhla ngolonwabo ngomso!
Ukubonelela nge-OEMChina Taq DNA Polymerase, Qpcr, Ngoku, sinika abathengi ngokusemthethweni izisombululo zethu eziphambili Kwaye ishishini lethu alikho kuphela "ukuthenga" kunye "nokuthengisa", kodwa ligxininisa ngakumbi.Sijonge ukuba ngababoneleli bakho abathembekileyo kunye nentsebenziswano yexesha elide eTshayina.Ngoku, sinethemba lokuba singabahlobo nawe.

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 bePrimer: 18-30bp.

Umxholo weGC: 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.

Ii-primers zoyilo kwindawo yesibini yesakhiwo setemplate kufuneka igwenywe kangangoko kunokwenzeka.

Gwema ukubunjwa kweziseko ezi-2 okanye ngaphezulu ezihambelanayo phakathi kwee-3′ zeziphelo ze-primers ezinyukayo kunye nezantsi.

Isiseko se-Primer 3′ asikwazi ukubakho nge-3 eyongezelelweyo elandelelanayo i-G okanye i-C.

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

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

ISihlomelo 1: Ipakethi yecandelo leCell Direct RT-qPCR Kit

1.Iseli Lysis Solution