Takhala wopanga odziwa.Kupambana ambiri paziphaso zofunikira pamsika wake pakuchotsera kwakukulu China High Sensitivity One-Step Probe Rt-QpcrKit V2, Ubwino ndi moyo wa fakitale, Yang'anani pazofuna zamakasitomala ndiye gwero la kupulumuka ndi chitukuko cha kampani, Timatsatira kukhulupirika ndi mtima wabwino wogwira ntchito, tikuyembekezera kubwera kwanu!
Takhala wopanga odziwa.Kupambana ambiri paziphaso zofunika kwambiri pamsika wakeChina Taq DNA Polymerase, Qpcr, Kampani yathu imalonjeza: mitengo yololera, nthawi yochepa yopanga ndi ntchito yokhutiritsa pambuyo pogulitsa, tikukulandiraninso kuti mudzayendere fakitale yathu nthawi iliyonse yomwe mukufuna.Ndikukhumba tsopano tili ndi bizinesi yosangalatsa komanso yayitali limodzi !!!

Kufotokozera

Chidachi chimagwiritsa ntchito njira yapadera ya lysis buffer yomwe imatha kumasula mwachangu RNA kuchokera ku ma cell otukuka pamachitidwe a RT-qPCR, potero amachotsa njira yoyeretsera RNA yomwe imatenga nthawi komanso yovuta.The RNA template ikhoza kupezeka mu mphindi 7 zokha.5 × Direct RT Mix ndi 2 × Direct qPCR Mix-SYBR reagents zoperekedwa ndi zida zitha kupeza mwachangu komanso moyenera zotsatira za PCR zenizeni zenizeni.

5 × Direct RT Mix ndi 2 × Direct qPCR Mix-SYBR ali ndi kulekerera kwamphamvu kwa inhibitor, ndipo ma lysate a zitsanzo angagwiritsidwe ntchito ngati template ya RT-qPCR mwachindunji.Chidachi chili ndi RNA yapamwamba kwambiri ya Foregene reverse transcriptase, ndi Hot D-Taq DNA polymerase, dNTPs, MgCl.₂, zotchingira zochita, PCR optimizer ndi stabilizer.

Zofotokozera

200×20μl Rxns, 1000×20μl Rxns

Zida zamagulu

Mbali & ubwino

■ Zosavuta komanso zothandiza : ndiukadaulo wa Cell Direct RT, zitsanzo za RNA zitha kupezeka m'mphindi 7 zokha.

■ Zofuna zachitsanzo ndizochepa, mpaka ma cell 10 akhoza kuyesedwa.

■ Kutulutsa kwakukulu: kumatha kuzindikira mwachangu RNA m'maselo opangidwa mu 384, 96, 24, 12, 6-chitsime mbale.

■ DNA chofufutira akhoza kuchotsa mwamsanga matupi athu omasulidwa, kwambiri kuchepetsa zotsatira zoyeserera.

■ Makina okhathamiritsa a RT ndi qPCR amapangitsa kuti masitepe awiri a RT-PCR alembedwe bwino kwambiri komanso PCR yachindunji, komanso yosagwirizana ndi RT-qPCR reaction inhibitors.

Kit application

Kuchuluka kwa ntchito: maselo otukuka.

- RNA yotulutsidwa ndi zitsanzo za lysis: imagwira ntchito pa template ya RT-qPCR ya zida izi.

- Zidazi zitha kugwiritsidwa ntchito pazifukwa izi: kusanthula kwa jini, kutsimikizira kwa siRNA-mediated gene silence effect, kuyang'anira mankhwala, etc.

Chithunzi

Kusungirako ndi moyo wa alumali

Gawo I la zida izi ziyenera kusungidwa pa 4 ℃;Gawo II liyenera kusungidwa pa -20 ℃.

Foregene Protease Plus II iyenera kusungidwa pa 4 ℃, osazizira pa -20 ℃.

Reagent 2 × Direct qPCR Mix-SYBR iyenera kusungidwa pa -20 ℃ mumdima;ngati amagwiritsidwa ntchito kawirikawiri, amathanso kusungidwa pa 4 ℃ kuti asungidwe kwakanthawi kochepa (ntchito mkati mwa masiku 10) .Kupambana ambiri paziphaso zofunikira za msika wake pakuchotsera kwakukulu China High Sensitivity One-Step Probe Rt-Qpcr Kit V2, Ubwino ndi moyo wa fakitale, Yang'anani pakufuna kwamakasitomala ndiye gwero la kupulumuka ndi chitukuko cha kampani, Timatsatira kukhulupirika ndi mtima wabwino wogwira ntchito, tikuyembekezera kubwera kwanu!
Kuchotsera kwakukuluChina Taq DNA Polymerase, Qpcr, Kampani yathu imalonjeza: mitengo yololera, nthawi yochepa yopanga ndi ntchito yokhutiritsa pambuyo pogulitsa, tikukulandiraninso kuti mudzayendere fakitale yathu nthawi iliyonse yomwe mukufuna.Ndikukhumba tsopano tili ndi bizinesi yosangalatsa komanso yayitali limodzi !!!

QuickEayiTM Cell Direct RT-qPCR Kit -Taqman

Cat.No.DRT-01021/01022

Kwa cell yolunjika RT-qPCR pogwiritsa ntchito ≤ 1000,000 ma cell

Chiyambi cha malonda

Izi zimagwiritsa ntchito njira yapadera ya lysis buffer kuti itulutse mwamsanga RNA kuchokera ku maselo opangidwa ndi chikhalidwe cha RT-qPCR, kuthetsa nthawi yowonongeka ndi yolemetsa ya RNA, ndi mphindi 7 zokha kuti mupeze template yofunikira ya RNA, ndi 5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman yoperekedwa ndi zida za nthawi yeniyeni ya PCR.

5 × Direct RT Mix ndi 2 × Direct qPCR Mix-Taqman ali ndi kulekerera kwamphamvu kwa inhibitor ndipo amatha kuchita bwino kusinthika ndi kukulitsa kwachindunji pogwiritsa ntchito lysate yachitsanzo kuti chiyezedwe ngati template.The reagent ili ndi Foregene Reverse Transcriptase, Hot D-Taq DNA Polymerase, dNTPs, MgCl.₂, Reaction Buffer, PCR Optimizer ndi Stabilizer, yomwe ingagwiritsidwe ntchito ndi lysis buffer kuti izindikire mwamsanga komanso mosavuta zitsanzo, ndipo imakhala ndi makhalidwe apamwamba, kukhazikika komanso kukhazikika.

Zogulitsa

Tekinoloje yosavuta, yothandiza ya Cell Direct RT yomwe imatenga mphindi 7 kuti mupeze zitsanzo za RNA.

Zofunikira zachitsanzo ndizochepa, ndipo maselo osachepera 10 otukuka angagwiritsidwe ntchito poyesera.

Kutulutsa kwakukulu kwa RNA kupeza mwachangu ma cell otukuka monga 384, 96, 24, 12, ndi 6-chitsime mbale.

DNA Eraser imatha kuchotsa mwachangu ma genome omasulidwa, kuchepetsa kwambiri zotsatira zoyeserera.

Makina okhathamiritsa a RT ndi qPCR amathandizira masitepe awiri a RT-PCR yokhala ndi zolembera zosinthika bwino, zatsatanetsatane, komanso kulolerana kwamphamvu kwa RT-qPCR reaction inhibitor.

Kit application

Kuchuluka kwa ntchito: Ma cell otukuka.

Zitsanzo za lysis zotanthauziridwa RNA: zimagwiritsidwa ntchito ngati template ya RT-qPCR ya magawo awiri.

Zida zitha kugwiritsidwa ntchito pazifukwa izi: kusanthula kwa gene regulatory expression, kuyesa kwa allele, kuyesa mankhwala, etc.

Kulephera kwa zida

Zidutswa zokulitsa ≤ 300 bp.

Zida zimagwiritsidwa ntchito popanga ma cell atsopano.

Kuwongolera khalidwe la mankhwala

Malinga ndi FOREGENE's Total Quality Management System, gulu lililonse la zida za Cell Direct RT-qPCR zimayesedwa mwamphamvu kangapo kuti zitsimikizire kudalirika komanso kukhazikika kwamtundu uliwonse wa zida.

Zamkatimu

*:Cell Lysis, 5×Direct RT Mix, 2× Direct qPCR Mix-Taqman zitha kugulidwa padera, zambiri zaperekedwa mu Appendix 1 (TSAMBA 13).

Zosungirako

1. Mikhalidwe Yotumizira

Njira yonse yotsika kutentha kwa ayezi pack box, kuonetsetsa kuti zida zili mu <4 °C state.

2. Zosungirako

Sungani gawo I pa 4°C ndi Gawo II pa -20°C.

The Foregene Protease Plus II iyenera kusungidwa pa 4°C, osati kuzizira pa -20°C.

Reagent 2 × Direct qPCR Mix-Taqman imasungidwa pa -20 ° C, kapena pa 4 ° C kuti igwiritsidwe ntchito kwakanthawi ngati imagwiritsidwa ntchito pafupipafupi (mkati mwa masiku 10).

Zambiri zagawo la Kit

Buffer CL: Imapereka malo ofunikira kuti ma cell lysis asinthe.

Buffer ST: Imathetsa zomwe zimagwira mu lysate kuti zipewe zotsatira za RT yotsatira.

DNA Eraser: DNA remover, zotsatira za kuchotsa genome pazoyeserera zotsatira.

5 × Direct RT Mix: Ili ndi kuyanjana kwapamwamba kwa RNA Foregene Reverse Transcriptase, RNase Inhibitor, dNTPs, stabilizers, enhancers, optimizers, ndi reverse transcript primer kuti igwirizane bwino (Random Primer, Oligo(dT)₁₈Choyamba).

Foregene Protease Plus II: Pankhani ya lysis buffer, maselo amapangidwa kuti atulutse nucleic acid.

2 × Direct qPCR Mix-Taqman: Reagent iyi ili ndi Hot D-Taq DNA Polymerase, dNTPs, MgCl₂, zosungira zochitira, PCR optimizer, ndi stabilizer.

20 × ROX Reference Dye: Nthawi zambiri amagwiritsidwa ntchito pa Real Time PCR zokulitsa zida za ABI, Stratagene ndi makampani ena, amagwiritsidwa ntchito kusintha kusiyana pakati pa machubu a PCR ndi machubu oyambitsidwa ndi zolakwika za PCR dosing.Kuphatikizika kwa 20 × ROX Reference Dye komwe kumafunikira pazida zosiyanasiyana ndikosiyana, ndipo wogwiritsa ntchito amatha kuwonjezera kutengera kuyika kwa chidacho.

RNase-Free ddH₂O: Madzi amtundu wa RNase opanda madzi oyera kwambiri pamachitidwe awiri a RT-qPCR.

Kusamalitsa:(Onetsetsani kuti mukuwerenga mosamala mosamala musanagwiritse ntchito zida)

Samalani njira yogwiritsira ntchito kuyesa kuti mupewe kuipitsidwa pakati pa zitsanzo.

Samalani ndi ukhondo wa malo oyesera ndi ziwiya kuti mupewe kuipitsidwa kwa RNase ndi kuwonongeka kwa RNA.

Tengani ma cell atsopano kapena osungidwa bwino ndipo musagwiritse ntchito ma cell omwe amaundana mobwerezabwereza.

5 × Direct qPCR Mix, 2 × Direct qPCR Mix-Taqman sayenera kuzizira mobwerezabwereza, apo ayi zingakhudze kumasulira kwapambuyo ndi PCR.

Prepachakudyakalentchito

Onetsetsani kuti mwawerenga malangizo mosamala musanagwiritse ntchito zidazi.Cell Direct RT-qPCR Kit ndi yosavuta, yosavuta, komanso yachangu kugwira ntchito, ndipo malangizowo amapereka chidziwitso chonse cha zida zonse ndi momwe angagwiritsire ntchito moyenera.Chonde konzani zofunikira zoyesera ndi zida musanagwiritse ntchito.

Zida zoyesera ndi zida

◆ Maselo a chikhalidwe.

◆ 1.5 ml kapena 2 ml, RNase-/DNase-Free centrifuge chubu, RNase-/DNase-Free nsonga, 0.2 ml wosabala qPCR chubu.

◆ qPCR makina, pipette, tabletop centrifuge (≥13,400×g) (malingana ndi zosowa zoyesera), ndi zina zotero.

Chitetezo

◆ Mankhwalawa ndi ofufuza zasayansi okha, chonde musagwiritse ntchito mankhwala, zachipatala, chakudya ndi zodzoladzola.

◆ Pogwiritsira ntchito mankhwala, valani zovala zoyenera za labu, magolovesi, magalasi otetezera, ndi zina zotero.

Ntchitootsogolera

Ma cell Lysis systems, RT systems, ndi qPCR reaction solution supplement packs zitha kugulidwa padera, kuti mudziwe zambiri mu Appendix 1 (TSAMBA 13).

Kalozera wa ntchito

A: Kutulutsidwa kwa RNA kwachitsanzo

1.Maselo adakonzedweratu: Tsukani mbale ya cell culture ndi PBS yozizira, kenako lyse maselo (10-10⁶), 10⁶ kuposa kuchuluka kwa maselo, tikulimbikitsidwa Foregene the Cell an RNA Isolation Kit the Total (DE-03111) kapena Animal Total RNA Isolation Kit (DE-03011) ya RNA m'zigawo ndi kuyeretsa.

1.1.Ma cell adherent (24- mbale ya chitsime monga chitsanzo)

1.1.1.Dziwani kuchuluka kwa ma cell pachitsime chilichonse, dziwani kuti ma cell ndi 1 × 10⁵, ndikugwiritsa ntchito pipette kuchotsa chikhalidwe cha chikhalidwe kuchokera ku mbale ya chikhalidwe.

1.1.2.Onjezani 200 μl wa 1 × PBS wokhazikika pa chitsime chilichonse.Osawombera mobwerezabwereza ndikuchotsa PBS m'zitsime.Pendekerani mbale ndikuchotsa PBS yochuluka momwe mungathere.Pitirizani ku sitepe 2.

1.1.3.Mbale yosiyana ya chikhalidwe cha ma cell kapena tebulo lachidziwitso 1-1 mu mbale yamtundu wa cell idawonjezedwa precooled 1 × PBS pakutsuka ma cell.

Table1-1: Mlingo wa PBS pama cell osiyanasiyana

Zindikirani:Kuonetsetsa olimba adherent maselo,chiwerengero chachikulu cha kutayika kwa maselo kumapewa pamene akutsuka .

1.2.Kuyimitsidwa maselo kapena omvera maselo otukuka mu mbale sanali porous

1.2.1.Maselo otsatizana otukuka m'mbale zopanda mipikisano bwino (maselo oyimitsidwa amayamba kuchokera ku sitepe yotsatira 1.2.2), sonkhanitsani ndikulekanitsa maselo molingana ndi njira yosonkhanitsira maselo, ndikuwayika mu mbale yachikhalidwe kapena chubu cha centrifuge;ngati trypsinization ntchito, amafuna centrifugation kusonkhanitsa maselo ndi kuchotsa trypsin otsalira, anawonjezera PBS resuspended maselo mu maselo munthu kumwazikana maselo.

1.2.2.Pambuyo chiwerengero cha maselo kuwerengedwa, aliquoted maselo 1 × 10⁵ imodzi ku machubu a centrifuge, sonkhanitsani maselo ndi centrifugation pa 1000 × g kwa 10 min.

1.2.3.Onjezani 200 μl PBS ku chubu cha centrifuge, musamapitirire mobwerezabwereza, ndikulakalaka PBS mwachindunji.pitirirani ku sitepe 2. (Ngati n'zovuta kutulutsa ndipo maselo adatsitsimutsidwa kachiwiri, akhoza kuchitidwa 1000 × g centrifuged 10 min mutataya supernatant, cell pellet ipitirire ku sitepe 2)

2.Cell lysis: Chotsani Buffer CL, kutentha kwake kumagwirizana ndi kutentha kwa chipinda, DNA Eraser ndi Foregene Protease Plus II, malinga ndi tebulo ili 1-2 lokonzekera dongosolo la lysis: (Lysis yankho lakonzeka kugwiritsidwa ntchito).

Table 1-2: cleavage kukonzekera dongosolo (Zindikirani: pokonzekera pa ayezi)

3. (24 -chitsime mbale monga chitsanzo) Pipette 200 μl wa cell lysis master mix mu chitsime chilichonse, Kuwomba mobwerezabwereza ka 5-10, Ikani kutentha kwa chipinda (20-25 ℃) kwa mphindi 5.

Zindikirani:Kupewa mapangidwe thovu, chonde pamene pipetting pipette lonse anasinthidwa 200μl kapena zochepa.Maselo amatha kuwoneka amtambo pambuyo pa lysis, zomwe ndi zachilendo.

4. (24 -mbale yabwino monga chitsanzo) imawonjezeredwa mumadzimadzi 20 μl Buffer ST (machitidwe osiyanasiyana a lysis Buffer ST anawonjezera mu ndalama zomwe zikuwonetsedwa mu Table 1-3), mobwerezabwereza pipetting 5-10 nthawi, kutentha kwa firiji (20-25 ℃) adayikidwa kwa 2 min.

Zindikirani:Nsonga ya pipette imayikidwa pansi pamtunda, kuonetsetsa kuti lysate yawonjezedwa,kupewa mapangidwe thovu, chonde pamene pipetting pipette lonse linasinthidwa 200μl kapena zochepa.

Gulu 1-3:Onjezani Buffer ST

5.Lysate imagwiritsidwa ntchito pazoyeserera za RT-qPCR.Ngati zoyeserera zotsatila sizingachitike munthawi yake, chonde sungani pa ayezi osapitilira 2hr, ndikusunga pa -20 ℃ kapena -80 ℃ (osapitilira miyezi itatu).

B: Kukonzekera kwa dongosolo la RT

1.Tulutsani 5 × Direct RT Mix ndikuyiyika pamadzi osambira, mulole kuti isungunuke mwachibadwa, ndikusakaniza mofatsa kuti mugwiritse ntchito;tulutsani RNase-Free ddH2O ndikuyisungunula ndikuyiyika pamadzi osambira kuti mudzagwiritse ntchito.Konzani kachitidwe ka ayezi molingana ndi Table 2-1 pansipa.

Table 2-1: Kukonzekera kwa RT reaction system

2.After akamaliza dongosolo chiphunzitso, mokoma wosanganiza ndi centrifuged mwachidule mu tebulo zotsatirazi 2 -2 anachita zinthu RT anachita.

Table 2-2: Kusintha kwa chikhalidwe cha RT Reaction

3.Atamaliza zomwe anachita, mankhwala anachita anayikidwa mwachindunji pa ayezi kwa qPCR , chonde ikani kusungidwa kwa nthawi yaitali -20 ℃ kapena -80 ℃ .

Zindikirani: Chifukwa chogwiritsa ntchito template yosayeretsedwa, madontho oyera atha kuwoneka m'mawu olembedwa mobwerera.Izi ndizochitika zachilendo.Centrifuge the supernatant yomweyo pazoyeserera zotsatila.

The chifukwa RT anachita njira anawonjezera kuti lotsatira Step Real Time PCR anachita kachitidwe, Ndi bwino kuwonjezera ndalama kuyambira 10-30% ya anachita dongosolo.

C: qPCR reaction system kukonzekera

1.Kuchuluka koyenera kwa B kukonzedwa mu sitepe ya cDNA template molingana ndi tebulo ili 3-1 kukonzekera kachitidwe kachitidwe.

Chidziwitso: Kuchuluka kwa template ya cDNA kumatenga 10-30% ya dongosolo la qPCR.Mwachitsanzo, mu dongosolo la 20μl qPCR, onjezani 2-6 μl ya lysis buffer, koma osapitirira 6 μl.

2.The kukhathamiritsa zinthu zabwino qPCR (Annealing kutentha, etc.) kwa qPCR anachita (The zinthu zimene anapatsidwa pa Table 3-2).

Chidziwitso: Yesani kugwiritsa ntchito mikhalidwe yokongoletsedwa ndi machitidwe a qPCR kuti mupeze zotsatira zabwino.

Table 3-1: Kukonzekera kwa PCR reaction system

1 *: Kukhazikika kwa primer kumatha kusinthidwa mumitundu ya 50-900 nM pomwe ntchito yoyambira imakhala yosauka.

Zindikirani: Dongosolo la qPCR litha kusinthidwa malinga ndi zosowa zoyeserera ndi mtundu wa cycler fluorescence.Kwa qPCR mu 50μl, sinthani mlingo wa reagent molingana ndi 20μl ndondomeko.

2 *: Sankhani koyenera komaliza kwa ROX Reference Dye malinga ndi fluorescence kuchuluka kwa matenthedwe cycler.Kuyika koyenera kwambiri kwa ROX Reference Dye pama cyclers wamba wa fluorescence akuwonetsedwa patebulo ili pansipa:

Table 3-2: Zochita za qPCR zimaperekedwa

Zindikirani: Kuti mupeze zotsatira zabwino kwambiri za qPCR, gradient PCR ingagwiritsidwe ntchito kukhathamiritsa momwe zimachitikira pama tempulo osiyanasiyana ndi zoyambira zosiyanasiyana.PCR anachita zinthu zimasiyanasiyana malinga ndi fluorescence analyzer, template, primer, etc. Mu ntchito yeniyeni, zinthu mulingo woyenera anachita ayenera kupangidwa malinga ndi zikhalidwe za fluorescence kuchuluka matenthedwe cycler, Chinsinsi mtundu, kukula kwa chidutswa cha chidwi, ndondomeko m'munsi mwa kaduka matalikidwe fragment ndi primers nthawi, ndi zina zotero.

Real Time PCR primer design mfundo

Forward Primer ndi Reverse Primer

Kwa Real Time PCR, mapangidwe oyambira ndi ofunika kwambiri.Zoyambira zimayenderana ndi kutsimikizika komanso kuthekera kwa kukulitsa kwa PCR, ndipo zitha kupangidwa motengera mfundo izi:

◆ Utali woyambira: 18-30bp.

◆ GC okhutira: 40-60%.

◆ Mtengo wa Tm: Mapulogalamu opangira mapulogalamu, monga Primer 5, akhoza kupereka mtengo wa Tm wa primer.Makhalidwe a Tm oyambira kumtunda ndi kumtunda ayenera kukhala pafupi momwe angathere.Njira yowerengera ya Tm ingagwiritsidwenso ntchito: Tm = 4 °C (G + C) + 2 °C (A + T).Pochita PCR, kutentha pansi pa mtengo woyambira wa Tm wa 5 ° C nthawi zambiri kumasankhidwa ngati kutentha kwapang'onopang'ono (kuwonjezeka kofananirako kwa kutentha kwapang'onopang'ono kumatha kukulitsa momwe PCR imachitira).

◆ Primers ndi zinthu za PCR:

◆ Design primer PCR amplification product kutalika makamaka 100-150bp.

◆ Zojambula zoyambira m'chigawo chachiwiri cha template ziyenera kupewedwa momwe zingathere.

◆ Pewani mapangidwe a 2 kapena zowonjezera zowonjezera pakati pa 3 'mapeto a mapiri okwera ndi otsika.

◆ Primer 3′ terminal base sangakhalepo ndi 3 owonjezera motsatizana G kapena C.

◆ Oyamba okhawo sangakhale ndi zida zowonjezera, apo ayi mawonekedwe a hairpin adzapangidwa, omwe amakhudza kukulitsa kwa PCR.

◆ ATCG iyenera kugawidwa mofanana momwe zingathere poyambira, ndipo 3′ terminal base iyenera kupewedwa monga T.

Zowonjezera1:Cndi DirectRT-qPCR Kit chigawot paketi yowonjezera

1.Cell Lysis Solution