Ukufumana ukwaneliseka kwabathengi yinjongo yenkampani yethu ngaphandle kwesiphelo.Siza kwenza iinzame ezimangalisayo zokuvelisa ukuthengisa okutsha kunye nomgangatho ophezulu, ukwanelisa iimfuno zakho ezizodwa kwaye sikubonelele ngokuthengiswa kwangaphambili, kwiintengiso kunye neenkonzo emva kokuthengisa zoMgangatho oPhezulu waseTshayina Universal Blue Sybr Green.Qpcr Master MixngeYellow Template Dilution Buffer, Sijolise ekuqhubeni ukuveliswa kwenkqubo, ukusungula ulawulo, ukusungula izinto ezintsha kunye nokuveliswa kweshishini, sinika umdlalo opheleleyo kwiinzuzo zizonke, kwaye ngokuqhubekayo siphucula inkonzo ekumgangatho ophezulu.
Ukufumana ukwaneliseka kwabathengi yinjongo yenkampani yethu ngaphandle kwesiphelo.Siza kwenza iinzame ezintle zokuvelisa ukuthengisa okutsha kunye nomgangatho ophezulu, ukwanelisa iimfuno zakho ezizodwa kwaye sikubonelele ngeenkonzo ezithengiswa kwangaphambili, ezithengiswayo kunye nasemva kokuthengisa.China Qpcr Premix, Qpcr Master Mix, Iwebhusayithi yethu yasekhaya yenze ngaphezulu kwe-50, 000 yeeodolo zokuthenga minyaka le kwaye iphumelele kakhulu ekuthengeni i-intanethi eJapan.Singakuvuyela ukufumana ithuba lokwenza ishishini nenkampani yakho.Ndijonge phambili ekufumaneni umyalezo wakho!

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 (ukusetyenziswa ngaphakathi kweentsuku ze-10) .Ukufumana ukwaneliseka kwabathengi yinjongo yenkampani yethu ngaphandle kokuphela.Siza kwenza iinzame ezimangalisayo zokuvelisa ukuthengisa okutsha kunye nomgangatho ophezulu, ukwanelisa iimfuno zakho ezizodwa kwaye sikubonelele ngokuthengiswa kwangaphambili, kwiintengiso kunye neenkonzo emva kokuthengisa zoMgangatho oPhezulu waseTshayina Universal Blue Sybr Green.Qpcr Master MixngeYellow Template Dilution Buffer, Sijolise ekuqhubeni ukuveliswa kwenkqubo, ukusungula ulawulo, ukusungula izinto ezintsha kunye nokuveliswa kweshishini, sinika umdlalo opheleleyo kwiinzuzo zizonke, kwaye ngokuqhubekayo siphucula inkonzo ekumgangatho ophezulu.
Umgangatho ophezuluChina Qpcr Premix, Qpcr Master Mix, Iwebhusayithi yethu yasekhaya iveliswe ngaphezulu kwe-50, 000 yeeodolo zokuthenga rhoqo ngonyaka kwaye iphumelele kakhulu ekuthengeni i-intanethi eJapan.Singakuvuyela ukufumana ithuba lokwenza ishishini nenkampani yakho.Ndijonge phambili ekufumaneni umyalezo wakho!

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