Mun kasance gogaggen masana'anta.Lashe mafi rinjaye a cikin mahimman takaddun shaida na kasuwa don babban ragi na China High Sensitivity Probe One-Mataki na Rt-QpcrKit V2, Inganci shine rayuwar masana'anta, Mayar da hankali kan buƙatun abokin ciniki shine tushen rayuwar kamfani da haɓakawa, Muna manne da gaskiya da yanayin aiki mai kyau, muna sa ran zuwan ku!
Mun kasance gogaggen masana'anta.Samun rinjaye a cikin mahimman takaddun shaida na kasuwar saChina Taq DNA Polymerase, Qpcr, Kamfaninmu yayi alkawalin: farashi mai ma'ana, gajeren lokacin samarwa da sabis na tallace-tallace mai gamsarwa, muna kuma maraba da ku don ziyarci masana'antar mu a kowane lokaci da kuke so.Fata yanzu muna da kasuwanci mai daɗi da dogon zango tare !!!

Bayani

Wannan kit ɗin yana amfani da tsarin buffer na musamman wanda zai iya sakin RNA da sauri daga samfuran ƙwayoyin halitta don halayen RT-qPCR, ta haka ne ke kawar da tsarin tsarkakewar RNA mai cin lokaci da wahala.Ana iya samun samfurin RNA a cikin mintuna 7 kawai.5 × Direct RT Mix da 2 × Direct qPCR Mix-SYBR reagents wanda kit ɗin ke bayarwa na iya samun sakamako mai ƙima na PCR cikin sauri da inganci.

5 × Direct RT Mix da 2 × Direct qPCR Mix-SYBR suna da ƙarfin juriya mai hanawa, kuma ana iya amfani da lysate na samfuran azaman samfuri don RT-qPCR kai tsaye.Wannan kit ɗin ya ƙunshi keɓaɓɓen RNA high-affinity Foregene reverse transcriptase, da Hot D-Taq DNA polymerase, dNTPs, MgCl.₂, amsawa buffer, PCR ingantawa da stabilizer.

Ƙayyadaddun bayanai

200 × 20μl Rxns, 1000 × 20μl Rxns

Abubuwan Kit

Fasaloli & fa'idodi

■ Sauƙi kuma mai tasiri: tare da fasahar Cell Direct RT, ana iya samun samfuran RNA a cikin mintuna 7 kacal.

∎ Bukatar samfurin karami ne, kamar yadda za a iya gwada ƙananan sel guda 10.

∎ Babban abin da ake fitarwa: zai iya gano RNA da sauri a cikin sel waɗanda aka tsara a cikin faranti 384, 96, 24, 12, 6-rijiya.

∎ Gogewar DNA na iya saurin cire kwayoyin halittar da aka fitar, da rage tasirin sakamakon gwaji na gaba.

∎ Ingantaccen tsarin RT da qPCR yana sa jujjuyawar matakai biyu na RT-PCR mafi inganci kuma PCR ta fi ƙayyadaddun ƙayyadaddun bayanai, kuma mafi juriya ga masu hana amsawar RT-qPCR.

Kit aikace-aikace

Iyakar aikace-aikace: sel masu al'ada.

- RNA da aka saki ta samfurin lysis: kawai ana amfani da samfurin RT-qPCR na wannan kit.

- Za a iya amfani da kit ɗin don dalilai masu zuwa: nazarin maganganun kwayoyin halitta, tabbatar da tasirin sirna mai tsaka-tsaki na yin shiru, gwajin magunguna, da sauransu.

zane

Adana da Rayuwar Shelf

Ya kamata a adana sashe na I na wannan kit a 4 ℃;Ya kamata a adana Sashe na II a -20 ℃.

Foregene Protease Plus II yakamata a adana shi a 4 ℃, kar a daskare a -20 ℃.

Reagent 2 × Direct qPCR Mix-SYBR yakamata a adana shi a -20℃ a cikin duhu;idan ana amfani dashi akai-akai, ana iya adana shi a 4 ℃ don ajiyar ɗan gajeren lokaci (amfani da sama a cikin kwanaki 10) . Mun kasance masu sana'a.Wining the majority in the crucial certifications of its market for Big discounting China High Sensitivity One-Step Probe Rt-Qpcr Kit V2, Quality ne factory' rayuwa , Mayar da hankali a kan abokin ciniki' bukatar ne tushen kamfanin tsira da kuma ci gaban , Mu adhere to gaskiya da kuma mai kyau bangaskiya aiki hali , suna jiran zuwan ku !
Babban rangwameChina Taq DNA Polymerase, Qpcr, Kamfaninmu ya yi alkawarin: farashi masu dacewa, gajeren lokacin samarwa da sabis na tallace-tallace mai gamsarwa, muna kuma maraba da ku don ziyarci masana'antar mu a kowane lokaci da kuke so.Fata yanzu muna da kasuwanci mai daɗi da dogon zango tare !!!

QuickEasyTM Cell Direct RT-qPCR Kit -Taqman

Farashin DRT-01021/01022

Don tantanin halitta kai tsaye RT-qPCR ta amfani da sel ≤ 1000,000

Gabatarwar samfur

Wannan samfurin yana amfani da tsarin buffer na musamman don sakin RNA da sauri daga samfuran ƙwayoyin halitta don halayen RT-qPCR, yana kawar da tsarin tsarkakewar RNA mai cin lokaci da wahala, kuma kawai mintuna 7 don samun samfurin RNA da ake buƙata, tare da 5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman wanda kit ɗin ya bayar zai iya samun saurin PCR-lokaci na gaske.

5 × Direct RT Mix da 2 × Direct qPCR Mix-Taqman suna da ƙarfin juriya mai hanawa kuma suna iya yin ingantaccen juzu'i da ƙayyadaddun haɓakawa ta amfani da lysate na samfurin da za a auna azaman samfuri.Reagent ya ƙunshi Foregene Reverse Transcriptase, Hot D-Taq DNA Polymerase, dNTPs, MgCl₂, Reaction Buffer, PCR Optimizer da Stabilizer, wanda za'a iya amfani dashi tare da buffer lysis don sauri da sauƙi gano samfurori, kuma yana da halaye na babban hankali, ƙayyadaddun ƙayyadaddun da kwanciyar hankali.

Siffofin samfur

Sauƙaƙan, fasaha mai inganci Cell Direct RT wanda ke ɗaukar ɗan mintuna 7 don samun samfuran RNA.

Samfuran buƙatun ƙanana ne, kuma ana iya amfani da mafi ƙarancin ƙwayoyin halitta 10 don gwaji.

Babban kayan aiki don saurin siyan RNA na ƙwayoyin halitta kamar su 384, 96, 24, 12, da faranti 6-rijiya.

Eraser na DNA yana iya saurin cire kwayoyin halittar da aka fitar, yana rage tasirin sakamako na gwaji na gaba.

Ingantattun tsarin RT da qPCR suna ba da damar RT-PCR mataki-biyu tare da ingantaccen juzu'i, ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai, da ƙarfin jurewar amsawar RT-qPCR.

Kit aikace-aikace

Iyakar aikace-aikacen: Kwayoyin Al'ada.

Samfurin lysis ya fassara RNA: ana amfani dashi azaman samfuri na RT-qPCR mai matakai biyu kawai.

Za a iya amfani da kit ɗin don dalilai masu zuwa: nazarin ka'idojin ka'idojin kwayoyin halitta, gwajin allele, gwajin magunguna, da sauransu.

Iyakokin Kit

Abubuwan da aka haɓaka ≤ 300 bp.

Ana amfani da kit ɗin don sabbin ƙwayoyin al'adu.

Kula da ingancin samfur

Dangane da Tsarin Gudanar da Ingancin Ingancin na FOREGENE, kowane nau'i na na'urori na Sel Direct RT-qPCR ana gwada su sosai sau da yawa don tabbatar da aminci da kwanciyar hankali na kowane nau'in kit ɗin.

Abubuwan da ke ciki

*:Cell Lysis, 5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman ana iya siyan shi daban, ana ba da cikakkun bayanai a shafi na 1 (SHAfi na 13).

Yanayin ajiya

1. Yanayin jigilar kaya

Dukkanin tsarin jigilar fakitin kankara mai ƙarancin zafin jiki, don tabbatar da cewa kit ɗin yana cikin yanayin <4 °C.

2. Yanayin ajiya

Ajiye sashin I a 4°C da Sashi na II a -20°C.

Foregene Protease Plus II yakamata a adana shi a 4°C, ba daskararre a -20°C ba.

Reagent 2× Direct qPCR Mix-Taqman ana adana shi a -20°C, ko a 4°C don amfani na ɗan gajeren lokaci idan ana amfani dashi akai-akai (a cikin kwanaki 10).

Bayanin bangaren Kit

Buffer CL: Yana ba da yanayin da ake buƙata don halayen lysis cell.

Buffer ST: Yana ƙare abu mai aiki a cikin lysate don guje wa tasiri akan RT na gaba.

Goge DNA: Mai cire DNA, tasirin cire kwayoyin halitta akan gwaje-gwaje na gaba.

5 × Direct RT Mix: Ya ƙunshi babban alaƙar RNA Foregene Reverse Transcriptase, RNase Inhibitor, dNTPs, stabilizers, enhancers, optimizers, and back transcrip primers for the best alignment (Random Primer, Oligo(dT)₁₈Farko).

Foregene Protease Plus II: A cikin mahallin lysis buffer, sel suna lysed don sakin acid nucleic.

2× Direct qPCR Mix-Taqman: Wannan reagent ya ƙunshi Hot D-Taq DNA Polymerase, dNTPs, MgCl₂, Matsakaicin amsawa, PCR ingantawa, da stabilizer.

20 × ROX Reference Dye: Gabaɗaya ana amfani dashi akan kayan haɓakawa na PCR na ABI, Stratagene da sauran kamfanoni, ana amfani dashi don daidaita bambanci tsakanin bututun PCR da bututun da ke haifar da kurakuran dosing PCR.20 × ROX Reference Dye taro da ake buƙata don kayan aiki daban-daban ya bambanta, kuma mai amfani zai iya ƙara shi bisa ga shawarar ƙaddamar da kayan aikin.

RNase-Free ddH₂O: Ruwa mai tsaftataccen ruwan da ba shi da RNase don halayen RT-qPCR mataki biyu.

Matakan kariya:(Tabbatar karanta matakan tsaro a hankali kafin amfani da kit ɗin)

Kula da hanyar aiki na gwaji don kauce wa giciye tsakanin samfurori.

Kula da tsabtar muhallin gwaji da kayan aiki don guje wa gurɓatawar RNase da lalata RNA.

Ɗauki samfurin tantanin halitta sabo ko ingantaccen tsari kuma kar a taɓa yin amfani da samfuran tantanin da aka narke akai-akai.

5× Direct qPCR Mix,2× Direct qPCR Mix-Taqman ya kamata a guje wa daskare-narke mai maimaitawa, in ba haka ba zai shafi juzu'i da kuma ingancin PCR.

Preparabon abincikafinaiki

Tabbatar karanta umarnin a hankali kafin amfani da wannan kit ɗin.Kit ɗin Cell Direct RT-qPCR mai sauƙi ne, dacewa, kuma mai sauri don aiki, kuma umarnin yana ba da cikakken bayani game da duka kit ɗin da yadda ake amfani da shi daidai.Da fatan za a shirya mahimman kayan gwaji da kayan aiki kafin amfani.

Kayan gwaji da kayan aiki

◆ Kwayoyin al'adu.

◆ 1.5 ml ko 2 ml, RNase-/DNase-Free centrifuge tube, RNase-/DNase-Free tip, 0.2 ml bakararre qPCR tube.

◆ qPCR inji, pipette, tabletop centrifuge (≥13,400×g) (dangane da buƙatun gwaji), da sauransu.

Tsaro

◆ Wannan samfurin don dalilai ne na bincike na kimiyya kawai, don Allah kar a yi amfani da shi don magunguna, na asibiti, abinci da dalilai na kwaskwarima.

◆ Lokacin amfani da sinadarai, sanya tufafin lab da suka dace, safar hannu, gilashin kariya, da sauransu.

Aikijagorori

Za a iya siyan tsarin Cell Lysis, tsarin RT, da fakitin ƙarin bayani na qPCR daban, don cikakkun bayanai a shafi na 1 (SHAfi 13).

Jagoran aiki

A: Misalin sakin RNA

1.Cells aka pretreated: A wanke farantin al'ada cell tare da sanyi PBS, sa'an nan lyse da Kwayoyin (10-10).⁶), 10⁶ fiye da adadin ƙwayoyin sel, ana ba da shawarar Foregene Cell ɗin RNA Keɓewar Kit ɗin Jima'i (DE-03111) ko Dabbobin Jimillar RNA Warewa Kit (DE-03011) don hakar RNA da tsarkakewa.

1.1.Kwayoyin maƙwabta (24- faranti a matsayin misali)

1.1.1.Ƙayyade adadin ƙwayoyin da ke cikin kowace rijiya, ƙayyade cewa adadin sel shine 1 × 10⁵, da kuma amfani da pipette don cire al'adun gargajiya daga tasa na al'ada.

1.1.2.Ƙara 200 μl na pre-sanyi 1 × PBS zuwa kowace rijiya.Kada a yi ta pipet akai-akai kuma cire PBS daga rijiyoyin.karkatar da farantin kuma cire yawan PBS gwargwadon yiwuwa.Ci gaba zuwa mataki na 2.

1.1.3.Daban-daban tasa al'adun tantanin halitta ko tebur mai lamba 1-1 a cikin tasa al'adun tantanin halitta an ƙara precooled 1 × PBS don wanke tantanin halitta.

Table1-1: Matsakaicin PBS don lambobi daban-daban na sel

Lura:Don tabbatar da tsayayyen sel masu mannewa,yawan asarar tantanin halitta da ake gujewa lokacin wankewa.

1.2.Kwayoyin dakatarwa ko sel masu ɗorewa waɗanda aka al'adarsu a cikin faranti marasa porous

1.2.1.Kwayoyin da ke da alaƙa da aka haɓaka a cikin faranti marasa rijiyoyin da ba su da yawa (kwayoyin dakatarwa suna farawa daga mataki na gaba na 1.2.2), tattara da kuma rarraba sel bisa ga hanyar tattara tantanin halitta na al'ada, kuma sanya su a cikin farantin al'ada ko centrifuge tube;idan an yi amfani da trypsinization, yana buƙatar centrifugation don tattara sel kuma don cire ragowar trypsin, ƙara PBS da aka sake dakatar da sel cikin sel guda ɗaya don tarwatsa sel.

1.2.2.Bayan an ƙidaya adadin ƙwayoyin sel, sel aliquoted 1 × 10⁵ daya zuwa centrifuge tubes, tattara sel ta hanyar centrifugation a 1000 × g na 10 min.

1.2.3.Ƙara 200 μl PBS zuwa bututun centrifuge, kada a sake yin pipet akai-akai, kuma kai tsaye neman PBS.Ci gaba zuwa mataki na 2. (Idan yana da wahalar hazo kuma an sake dakatar da sel, ana iya yin 1000×g a tsakiya na minti 10 bayan watsar da mai girma, pellet tantanin halitta ya ci gaba zuwa mataki na 2)

2.Cell lysis: Cire Buffer CL, yawan zafin jiki ya daidaita zuwa zafin jiki, DNA Eraser da Foregene Protease Plus II, bisa ga tebur mai zuwa 1-2 da aka shirya tsarin lysis: (Maganin Lysis yana shirye don amfani).

Tebur 1-2: tsagewa shirye-shiryen tsarin (Lura: a cikin shirye-shiryen akan kankara)

3.( 24 -well farantin a matsayin misali) Pipette 200 μl na cell lysis master mix a cikin kowace rijiya, akai-akai busa 5-10 sau, Incubate a dakin zafin jiki (20-25 ℃) for 5 min.

Lura:Don guje wa samuwar kumfa, don Allah lokacin da aka daidaita ma'aunin pipette pipette zuwa 200μl ko ƙasa da haka.Kwayoyin na iya bayyana gajimare bayan lysis, wanda yake al'ada.

4. (24 -well farantin a matsayin misali) an kara a cikin ruwa 20 μl Buffer ST ( daban-daban lysis tsarin Buffer ST kara a cikin adadin da aka nuna a cikin Table 1-3), maimaita pipetting 5-10 sau, a dakin zafin jiki (20-25 ℃) An incubated for 2 min.

Lura:Tushen pipette da aka zubar a ƙasa da ƙasa, yana tabbatar da cewa an ƙara lysate,don kaucewa samuwar kumfa, don Allah lokacin da aka daidaita ma'aunin pipette pipette zuwa 200μl ko ƙasa da haka.

Table 1-3:Ƙara Buffer ST

5.An yi amfani da lysate don gwaje-gwajen RT-qPCR na gaba.Idan ba za a iya yin gwaje-gwajen na gaba a cikin lokaci ba, da fatan za a ajiye shi a kan kankara don kada ya wuce 2hr, kuma adana a -20 ℃ ko -80 ℃ (ba fiye da watanni uku ba).

B: RT tsarin shirye-shiryen

1.Ka fitar da 5 × Direct RT Mix kuma sanya shi a kan wanka mai kankara, bar shi ya narke, kuma a hankali a haɗa shi don amfani daga baya;fitar da ddH2O-Free RNase a narke shi kuma sanya shi a kan wankan kankara don amfani daga baya.Shirya tsarin amsawa akan kankara bisa ga Table 2-1 da ke ƙasa.

Table 2-1: Shiri na RT dauki tsarin

2.Bayan kammala tsarin tsarin, a hankali gauraye da centrifuged a takaice a cikin tebur mai zuwa 2 -2 halayen halayen RT.

Table 2-2: RT Reaction yanayin saitin

3.Bayan kammala amsawa, an sanya samfurin samfurin kai tsaye a kan kankara don qPCR, don Allah sanya dogon lokaci kiyayewa -20 ℃ ko -80 ℃.

Lura: Saboda amfani da samfurin da ba a tsarkake ba, farin hazo na iya bayyana a cikin samfurin rubutun baya.Wannan lamari ne na al'ada.Centrifuge supernatant nan da nan don gwaje-gwaje na gaba.

Sakamakon sakamako na RT an ƙara shi zuwa tsarin amsawa na gaba na Real Time PCR, ana ba da shawarar ƙara adadin adadin daga 10-30% na tsarin amsawa.

C: qPCR dauki tsarin shiri

1.Ya dace adadin B shirya a mataki cDNA samfuri bisa ga tebur mai zuwa 3-1 don shirya tsarin amsawa.

Lura: Adadin samfurin cDNA yana lissafin 10-30% na tsarin qPCR.Misali, a cikin tsarin 20μl qPCR, ƙara 2-6 μl na buffer lysis, amma bai wuce 6 μl ba.

2.The ingantawa mai kyau qPCR yanayi (Annealing zafin jiki, da dai sauransu) domin qPCR dauki (The dauki yanayi bayar a Table 3-2).

Lura: Gwada amfani da ingantattun yanayi don halayen qPCR don samun kyakkyawan sakamako.

Table 3-1: Shiri na PCR dauki tsarin

1 *: Za'a iya daidaita maida hankali na farko a cikin kewayon 50-900 nM lokacin da aikin amsawa ya kasance mara kyau.

Lura: Ana iya daidaita tsarin qPCR bisa ga buƙatun gwaji da ƙirar cycler fluorescence.Don qPCR a cikin 50μl tsarin, daidaita adadin reagent daidai gwargwado bisa ga 20μl tsarin.

2*: Zaɓi daidaitaccen taro na ƙarshe na ROX Reference Dye bisa ga ma'aunin zafi mai zafi.Mafi dacewa da ROX Reference Reference maida hankali ga gama gari masu yin cycler fluorescence ana nuna su a cikin tebur da ke ƙasa:

Tebur 3-2: An ba da yanayin halayen qPCR

Lura: Domin samun mafi kyawun tasirin qPCR, ana iya amfani da PCR gradient don inganta yanayin amsawa don samfura daban-daban da maɓalli daban-daban.PCR dauki yanayi bambanta dangane da kyalli analyzer, samfuri, firamare, da dai sauransu A cikin takamaiman aiki, da mafi kyau duka dauki yanayi bukatar da za a tsara bisa ga takamaiman yanayi na kyalli quantitative thermal cycler, samfur nau'in, girman da guntu na sha'awa, tushe jerin karawa gutsuttsura da GC abun ciki da kuma tsawon lokaci da zafin jiki da dai sauransu, ciki har da wani lokaci zazzabi, da dai sauransu

Ka'idodin ƙira na ainihin lokacin PCR

Gaba Farawa da Juya Farko

Don Real Time PCR, ƙirar farko tana da mahimmanci.Abubuwan haɓaka suna da alaƙa da ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙimar PCR, kuma ana iya tsara su tare da la'akari da ƙa'idodi masu zuwa:

◆ Tsawon farko: 18-30bp.

◆ GC abun ciki: 40-60%.

◆ Darajar Tm: Software na ƙira na farko, irin su Primer 5, na iya ba da ƙimar Tm na farkon.Ma'auni na Tm na sama da na ƙasa ya kamata su kasance kusa da iyawa.Hakanan ana iya amfani da dabarar lissafin Tm: Tm = 4 °C (G + C) + 2 °C (A + T).Lokacin yin PCR, ana zaɓar zafin da ke ƙasa da ƙimar ƙimar Tm na 5 ° C gabaɗaya azaman zazzabi mai ɗaukar nauyi (madaidaicin haɓakar zafin jiki na iya ƙara ƙayyadaddun halayen PCR).

◆ Alamar farko da samfuran PCR:

◆ Design primer PCR haɓaka samfurin tsawon ya fi dacewa 100-150bp.

◆ Za a kauce wa abubuwan da aka tsara a cikin yanki na biyu na samfuri gwargwadon yiwuwa.

◆ Guji samuwar tushe guda 2 ko sama da haka tsakanin 3′ na ƙofofin sama da na ƙasa.

◆ Primer 3′ tushe tushe ba zai iya kasancewa tare da ƙarin 3 a jere G ko C.

◆ Na'urorin da kansu ba za su iya samun ƙarin sifofi ba, in ba haka ba za a kafa tsarin gashin gashi, yana shafar haɓaka PCR.

◆ Ya kamata a rarraba ATCG daidai gwargwado kamar yadda zai yiwu a cikin jeri na farko, kuma ya kamata a guje wa tushe na 3′ a matsayin T.

Karin bayani1:Cda DirectRT-qPCR Kit component kari kunshin

1.Cell Lysis Magani