Anyị nwere ahụmahụ emeputa.Inweta ọtụtụ n'ime asambodo dị mkpa nke ahịa ya maka nnukwu mbelata China High Sensitivity One-Step Probe Rt-QpcrKit V2, Ogo bụ factory 'ndụ, Lekwasị anya na ndị ahịa' ina bụ isi iyi nke nlanarị ụlọ ọrụ na mmepe, Anyị na-agbaso eziokwu na ezi okwukwe na-arụ ọrụ àgwà, na-atụ anya gị na-abịa !
Anyị nwere ahụmahụ emeputa.Na-emeri ọtụtụ n'ime asambodo dị mkpa nke ahịa ya makaChina Taq DNA Polymerase, Qpcr, Ụlọ ọrụ anyị na-ekwe nkwa: ọnụ ahịa ezi uche dị na ya, obere oge mmepụta na ọrụ ahịa na-eju afọ, anyị na-anabatakwa gị ka ị gaa na ụlọ ọrụ anyị n'oge ọ bụla ịchọrọ.Achọrọ m ugbu a anyị nwere azụmahịa dị ụtọ na ogologo okwu ọnụ !!!

Nkọwa

Ngwa a na-eji sistemu nchekwa lysis pụrụ iche nke nwere ike hapụ RNA ngwa ngwa site na nlele cell emebere maka mmeghachi omume RT-qPCR, si otú a na-ewepụ usoro ịdị ọcha nke RNA na-ewe ma na-arụsi ọrụ ike.Enwere ike nweta ndebiri RNA n'ime naanị nkeji 7.Ngwakọta 5 × Direct RT Mix na 2 × Direct qPCR Mix-SYBR reagents nke ngwa ahụ na-enye nwere ike nweta nsonaazụ PCR ozugbo na nke ọma.

5 × Direct RT Mix na 2 × Direct qPCR Mix-SYBR nwere nnabata inhibitor siri ike, enwere ike iji lysate nke ihe nlele ahụ dị ka ndebiri maka RT-qPCR ozugbo.Ngwa a nwere RNA pụrụ iche Foregene reverse transcriptase, yana Hot D-Taq DNA polymerase, dNTPs, MgCl.₂, ihe nchekwa mmeghachi omume, PCR optimizer na stabilizer.

Nkọwapụta

200 × 20μl Rxns, 1000 × 20μl Rxns

Ngwa ngwa

Atụmatụ & uru

∎ Dị mfe ma dị irè: site na teknụzụ Cell Direct RT, enwere ike nweta ihe nlele RNA n'ime naanị nkeji asaa.

■ Ọchịchọ nlele dị ntakịrị, ebe ọ bụ na enwere ike ịnwale mkpụrụ ndụ 10.

∎ Mgbakwunye dị elu: ọ nwere ike ịchọpụta RNA ngwa ngwa n'ime sel ndị emebere na 384, 96, 24, 12, 6-well plates.

∎ DNA Eraser nwere ike wepụ ngwa ngwa genomes ewepụtara, belata mmetụta dị na nsonaazụ nnwale na-esote.

∎ Sistemu RT na qPCR emezigharịrị na-eme ka ntụgharị ntụgharị RT-PCR nzọụkwụ abụọ rụọ ọrụ nke ọma yana PCR akọwapụtara nke ọma, ma na-eguzogide ndị na-egbochi mmeghachi omume RT-qPCR.

Ngwa ngwa

Oke ngwa: mkpụrụ ndụ omenala.

- RNA wepụtara site na nlele lysis: naanị ọdabara na ndebiri RT-qPCR nke ngwa a.

- Enwere ike iji ngwa ahụ maka ebumnuche ndị a: nyocha okwu mkpụrụ ndụ ihe nketa, nkwenye nke mmetụta ịgbachitere mkpụrụ ndụ ihe nke siRNA, nyocha ọgwụ, wdg.

Eserese

Nchekwa na Shelf ndụ

A ga-echekwa akụkụ nke mbụ nke ngwa a na 4 ℃;Ekwesịrị ịchekwa akụkụ II na -20 ℃.

Ekwesịrị ịchekwa Foregene Protease Plus II na 4 ℃, echekwala na -20 ℃.

Ekwesịrị ịchekwa Reagent 2 × Direct qPCR Mix-SYBR na -20℃ n'ọchịchịrị;ma ọ bụrụ na-eji ugboro ugboro, ọ nwekwara ike-echekwara na 4 ℃ maka obere oge nchekwa (jiri elu n'ime 10 ụbọchị) .Anyị nwere ahụmahụ emeputa.Wining the most in the crucial certifications of its market for Big discounting China High Sensitivity One-Step Probe Rt-Qpcr Kit V2, Quality is factory' life , Focus on customers' demand is the source of company survival and development , We adhere to honesty and good faith working behavior, looking forward to your coming !
Nnukwu mbelataChina Taq DNA Polymerase, Qpcr, Ụlọ ọrụ anyị na-ekwe nkwa: ọnụ ahịa ezi uche dị na ya, oge mmepụta obere oge na ọrụ na-enye afọ ojuju mgbe ịre ahịa, anyị na-anabatakwa gị ka ị gaa na ụlọ ọrụ anyị n'oge ọ bụla ịchọrọ.Achọrọ m ugbu a anyị nwere azụmahịa dị ụtọ na ogologo okwu ọnụ !!!

QuickEasyTM Cell Direct RT-qPCR Kit -Taqman

Cat.No.DRT-01021/01022

Maka cell kpọmkwem RT-qPCR na-eji sel ≤ 1000,000

Ntinye ngwaahịa

Ngwaahịa a na-eji sistemụ nchekwa lysis pụrụ iche iji wepụta RNA ngwa ngwa site na ihe nlele cell emebere maka mmeghachi omume RT-qPCR, na-ewepụ usoro ịdị ọcha nke RNA na-eri oge na nke siri ike, yana naanị nkeji 7 iji nweta ndebiri RNA achọrọ, yana 5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman nyere site na ngwa ahụ nwere ike nweta ngwa ngwa PCR na ezigbo oge.

5 × Direct RT Mix na 2 × Direct qPCR Mix-Taqman nwere nnabata inhibitor siri ike ma nwee ike ịrụ ọrụ ntụgharị nke ọma na nkwalite akọwapụtara site na iji lysate nke ihe atụ a ga-atụle dị ka ndebiri.Reagent nwere Foregene Reverse Transcriptase, Hot D-Taq DNA Polymerase, dNTPs, MgCl.₂, Reaction Buffer, PCR Optimizer na Stabilizer, nke enwere ike iji ya na lysis buffer iji chọpụta ngwa ngwa ngwa ngwa, ma nwee njirimara nke mmetụta dị elu, nkọwa na nkwụsi ike.

Njirimara ngwaahịa

Teknụzụ Cell Direct RT dị mfe, dị irè nke na-ewe ihe dị ka nkeji 7 iji nweta nlele RNA.

Ihe nlele chọrọ dị ntakịrị, enwere ike iji opekempe sel iri emebere maka nnwale.

Nbudata dị elu maka inweta ngwa ngwa RNA nke mkpụrụ ndụ emebere dịka 384, 96, 24, 12, na 6-well plates.

Nchicha DNA na-enwe ike iwepụ ngwa ngwa genomes ewepụtara, na-ebelata mmetụta dị na nsonaazụ nnwale na-esote.

Sistemụ RT na qPCR kachasị mma na-eme ka RT-PCR nzọụkwụ abụọ nwee ntụgharị ntụgharị nke ọma karịa, nkọwapụta, yana nnabata ihe mgbochi mmeghachi omume RT-qPCR siri ike.

Ngwa ngwa

Oke ngwa: mkpụrụ ndụ mebere.

Sample lysis tụgharịrị RNA: ejiri naanị dị ka ndebiri RT-qPCR nzọụkwụ abụọ.

Enwere ike iji ngwa maka ebumnuche ndị a: nyocha okwu usoro iwu mkpụrụ ndụ ihe nketa, ule allele, nyocha ọgwụ, wdg.

Oke ngwa

Iberibe agbakwunyere ≤ 300 bp.

A na-eji ngwa eme ihe na mkpụrụ ndụ omenala ọhụrụ.

njikwa mma ngwaahịa

Dị ka FOREGENE's Total Quality Management System si dị, a na-anwale ngwa ngwa Cell Direct RT-qPCR ọ bụla nke ọma ọtụtụ oge iji hụ na ntụkwasị obi na nkwụsi ike nke ogo ngwa ngwa ọ bụla.

Ngwa ọdịnaya

*:Cell Lysis, 5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman nwere ike zụta iche, enyere nkọwa na mgbakwunye 1 (PAGE 13).

Ọnọdụ nchekwa

1. Ọnọdụ mbupu

Usoro dum nke ụgbọ njem igbe ice dị ala, iji hụ na ngwa ahụ dị na steeti <4 °C.

2. Ọnọdụ nchekwa

Chekwaa akụkụ I na 4 Celsius C yana akụkụ II na -20 Celsius.

Ekwesịrị ịchekwa Foregene Protease Plus II na 4°C, ọ bụghị oyi kpọnwụrụ na -20°C.

A na-echekwa Reagent 2 × Direct qPCR Mix-Taqman na -20°C, ma ọ bụ na 4°C maka ojiji nwa oge ma ọ bụrụ na ejiri ya ugboro ugboro (n'ime ụbọchị iri).

Ozi akụrụngwa akụrụngwa

Buffer CL: Na-enye gburugburu ebe achọrọ maka mmeghachi omume lysis cell.

Buffer ST: Na-akwụsị ihe na-arụ ọrụ na lysate iji zere mmetụta na RT na-esote.

Nchicha DNA: Ihe na-ewepụ DNA, mmetụta nke iwepu genome na nnwale ndị ọzọ.

Ngwakọta 5 × Direct RT: nwere njikọ RNA dị elu Foregene Reverse Transcriptase, RNase Inhibitor, dNTPs, stabilizers, enhancers, optimizers, and reverse transcription primers maka nhazi kacha mma (Random Primer, Oligo(dT)₁₈Ihe mbụ).

Foregene Protease Plus II: N'ihe gbasara lysis buffer, mkpụrụ ndụ na-lysed ka ha wepụta nucleic acid.

2 × Direct qPCR Mix-Taqman: Nke a reagent nwere Hot D-Taq DNA Polymerase, dNTPs, MgCl₂, ihe nchekwa mmeghachi omume, PCR optimizer, na stabilizer.

20 × ROX Reference Dye: A na-ejikarị na Real Time PCR amplification instruments nke ABI, Stratagene na ụlọ ọrụ ndị ọzọ, a na-eji ya dozie ọdịiche dị n'etiti tubes PCR na tubes kpatara PCR dosing njehie.Ntụle 20 × ROX Reference Dye nke achọrọ maka ngwa dị iche iche dị iche iche, onye ọrụ nwere ike ịgbakwunye ya dịka ntinye nke akụrụngwa akwadoro.

ddH na-efu RNase₂O: mmiri dị ọcha sterilized na-enweghị RNase maka mmeghachi omume RT-qPCR nzọụkwụ abụọ.

Mkpachapụ anya:(Jide n'aka na-agụọ nlezianya anya tupu i jiri ngwa ahụ)

Lezienụ anya na usoro ọrụ nke nnwale ahụ iji zere mmetọ n'etiti ihe atụ.

Lezienụ anya na ịdị ọcha nke gburugburu nnwale na arịa iji zere mmetọ RNase na mmebi RNA.

Were ihe nlele cell ọhụrụ ma ọ bụ nke echekwara nke ọma ma ghara iji ihe nlele cell gbazere ugboro ugboro.

5 × Direct qPCR Mix, 2 × Direct qPCR Mix-Taqman kwesịrị izere ịza oyi ugboro ugboro, ma ọ bụghị ya, ọ ga-emetụta ntụgharị ntụgharị na arụmọrụ PCR.

Nkwadebenri nrimbụọrụ

Jide n'aka na-agụ ntuziaka ahụ nke ọma tupu i jiri ngwa a.Kit Cell Direct RT-qPCR dị mfe, adaba na ngwa ngwa iji rụọ ọrụ, ntuziaka ahụ na-enye ozi zuru oke gbasara ngwa ahụ yana otu esi eji ya nke ọma.Biko kwadebe ihe nnwale na akụrụngwa dị mkpa tupu ejiri ya.

Ngwa na akụrụngwa nnwale

◆ Mkpụrụ ndụ omenala.

◆ 1.5 ml ma ọ bụ 2 ml, RNase-/DNase-Free centrifuge tube, RNase-/DNase-Free n'ọnụ, 0.2 ml qPCR tube sterile.

◆ qPCR igwe, pipette, tabletop centrifuge (≥13,400×g) (dabere na mkpa nnwale), wdg.

Nchekwa

◆ Ngwaahịa a bụ naanị maka nyocha sayensị, biko ejila ya maka ọgwụ, ụlọ ọgwụ, nri na ebumnuche ịchọ mma.

◆ Mgbe ị na-eji kemịkalụ, yi uwe ụlọ nyocha kwesịrị ekwesị, uwe aka, ugegbe nchebe, wdg.

Ọrụndu

Enwere ike ịzụta sistemu Cell Lysis, sistemu RT na qPCR mmeghachi omume mmeju nke dị iche iche, maka nkọwa na mgbakwunye 1 (PAGE 13).

Ntuziaka ọrụ

A: Ntọhapụ RNA atụ

1.Cells e pretreated: Saa na cell omenala efere na oyi na-atụ PBS, wee lyse cell (10-10).⁶), 10⁶ karịa ọnụ ọgụgụ nke mkpụrụ ndụ, a na-atụ aro Foregene the Cell an RNA Isolation Kit Total (DE-03111) ma ọ bụ Animal Total RNA Isolation Kit (DE-03011) maka mmịpụta RNA na ịdị ọcha.

1.1.Mkpụrụ ndụ na-ejikọta ya (ọkara 24 dị ka ihe atụ)

1.1.1.Kpebie ọnụọgụ mkpụrụ ndụ dị na olulu mmiri nke ọ bụla, chọpụta na ọnụọgụ mkpụrụ ndụ bụ 1 × 10⁵, ma jiri pipette wepụ omenala omenala na efere omenala.

1.1.2.Tinye 200 μl nke chilled 1 × PBS na olulu mmiri nke ọ bụla.Ejila pipet ugboro ugboro wee wepụ PBS na olulu mmiri.Tụgharịa efere ahụ ma wepụ PBS dị ka o kwere mee.Gaba na nzọụkwụ 2.

1.1.3.A na-etinye efere omenala cell dị iche iche ma ọ bụ tebụl nọmba ntụaka 1-1 n'ime efere omenala cell nke etinyechara 1 × PBS maka ịsacha cell.

Isiokwu 1-1: Usoro PBS maka ọnụọgụ dị iche iche nke sel

Rịba ama:Iji hụ na sel na-akwadosi ike,a na-ezere ọnụ ọgụgụ dị ukwuu nke ọnwụ cell mgbe ị na-asa .

1.2.Selụ nkwusioru ma ọ bụ sel ndị na-eso ụzọ emebere n'ime efere na-enweghị oghere

1.2.1.Mkpụrụ ndụ ndị na-ejikọta ya na-etolite na efere ndị na-abụghị nke ọma (mkpụrụ ndụ nkwusioru na-amalite site na nzọụkwụ ọzọ 1.2.2), na-anakọta ma kewaa mkpụrụ ndụ dịka usoro nchịkọta cell nkịtị si dị, ma tinye ha na efere omenala ma ọ bụ tube centrifuge;Ọ bụrụ na a na-eji trypsinization eme ihe, chọrọ centrifugation iji kpokọta mkpụrụ ndụ na iwepu trypsin fọdụrụnụ, gbakwunyere PBS kpọghachite sel n'ime sel nke ọ bụla iji gbasasịa sel.

1.2.2.Mgbe ọnụọgụ nke mkpụrụ ndụ gụchara, mkpụrụ ndụ aliquoted 1 × 10⁵ otu na tubes centrifuge, na-anakọta mkpụrụ ndụ site na centrifugation na 1000 × g maka 10 min.

1.2.3.Tinye 200 μl PBS na tube centrifuge, emela pipet ugboro ugboro, ma chọọ PBS ozugbo.gaa n'ihu na nzọụkwụ 2. (Ọ bụrụ na ọ siri ike ịmalite na sel ndị ahụ na-emegharị ọzọ, enwere ike ịme 1000 × g centrifuged 10 min mgbe ị tụfuru ihe dị elu, pellet cell na-aga n'ihu na nzọụkwụ 2)

2.Cell lysis: Wepụ Buffer CL, ọnọdụ okpomọkụ ya kwadoro na ọnụ ụlọ okpomọkụ, DNA Eraser na Foregene Protease Plus II, dị ka okpokoro na-esonụ 1-2 kwadebere lysis usoro: (Lysis ngwọta dị njikere maka ojiji).

Tebụl 1-2: mgbawa nkwadebe usoro (Rịba ama: na nkwadebe na ice)

3. (24 -well efere dị ka ihe atụ) Pipette 200 μl nke cell lysis master mix n'ime nke ọ bụla nke ọma, ugboro ugboro na-efe efe 5-10 ugboro, Incubate na ụlọ okpomọkụ (20-25 ℃) maka 5 min.

Rịba ama:Iji zere nguzobe nke afụ, biko mgbe a na-agbanwe ọnụ ọgụgụ pipette pipette ka ọ bụrụ 200μl ma ọ bụ obere.Selụ nwere ike ịpụta urukpuru mgbe lysis gasịrị, nke bụ ihe nkịtị.

4. (24 -well efere dị ka ihe atụ) na-agbakwunyere na mmiri mmiri 20 μl Buffer ST (dị iche iche usoro lysis Buffer ST agbakwunyere na ego gosiri na Tebụl 1-3), ugboro ugboro pipetting 5-10 ugboro, na ụlọ okpomọkụ (20-25 ℃) e incubated maka 2 min.

Rịba ama:Ngwunye pipette tụfuru n'okpuru ala, na-eme ka lysate tinye ya,iji zere nguzobe nke afụ, biko mgbe a na-agbanwe ọnụ ọgụgụ pipette pipette ka ọ bụrụ 200μl ma ọ bụ obere.

Isiokwu 1-3:Tinye ihe nchekwa ST

5.The lysate na-eji maka ụdi RT-qPCR nnwale.Ọ bụrụ na enweghị ike ịme nnwale ndị na-esote n'oge, biko debe ya na ice maka ihe karịrị awa 2, wee chekwaa na -20 ℃ ma ọ bụ -80 ℃ (karịa ọnwa atọ).

B: RT nhazi usoro

1.Wepụ 5 × Direct RT Mix ma tinye ya na ice bat, hapụ ya ka ọ gbazee ndammana, ma jiri nwayọọ gwakọta ya maka ojiji ọzọ;wepụta RNase-Free ddH2O wee gbazee ya ma tinye ya na bath ice maka iji mechaa.Kwadebe usoro mmeghachi omume na ice dị ka Tebụl 2-1 dị n'okpuru.

Tebụl 2-1: Nkwadebe nke usoro mmeghachi omume RT

2.Mgbe emechara usoro nhazi usoro, jiri nwayọọ gwakọta na centrifuged nkenke na tebụl na-esonụ 2 -2 mmeghachi omume mmeghachi omume RT.

Tebụl 2-2: Ntọala ọnọdụ mmeghachi omume RT

3.Mgbe emechara mmeghachi omume ahụ, a na-etinye ngwaahịa mmeghachi omume ozugbo na ice maka qPCR, biko tinye nchekwa ogologo oge -20 ℃ ma ọ bụ -80 ℃.

Mara: N'ihi iji ndebiri anaghị adị ọcha, mkpọda ọcha nwere ike ịpụta na ngwaahịa ntụgharị azụ.Nke a bụ ihe nkịtị.Centrifuge supernatant ozugbo maka nnwale ndị ọzọ.

A na-agbakwunye azịza nzaghachi RT nke na-esote na usoro mmeghachi omume PCR nke na-esote, a na-atụ aro ka ịgbakwunye ọnụọgụ sitere na 10-30% nke usoro mmeghachi omume.

C: qPCR mmeghachi omume nkwadebe

1. Kwesịrị ekwesị ego nke B kwadebere na nzọụkwụ cDNA template dị ka ndị na-esonụ table 3-1 iji kwadebe a mmeghachi omume usoro.

Mara: Ọnụ ego template cDNA bụ 10-30% nke sistemu qPCR.Dịka ọmụmaatụ, na sistemụ 20μl qPCR, tinye 2-6 μl nke nchekwa lysis, mana ọ bụghị karịa 6 μl.

2.The njikarịcha mma qPCR ọnọdụ (Annealing okpomọkụ, wdg) maka qPCR mmeghachi omume (The mmeghachi omume nyere na Table 3-2).

Mara: Gbalịa iji ọnọdụ kachasị maka mmeghachi omume qPCR iji nweta nsonaazụ kacha mma.

Tebụl 3-1: Nkwadebe nke usoro mmeghachi omume PCR

1 *: Enwere ike ịhazi ntinye uche nke mbụ na oke nke 50-900 nM mgbe ọrụ mmeghachi omume primer adịghị mma.

Mara: Enwere ike idozi sistemu qPCR dabere na mkpa nnwale na ụdị cycler fluorescence.Maka qPCR na 50μl Sistemu, mezie reagent onunu ogwu nke kwesiri ka 20μl sistemu.

2 *: Họrọ ntinye ikpeazụ kwesịrị ekwesị nke ROX Reference Dye dị ka fluorescence quantitative thermal cycler si dị.Ntụle ntụaka ntụaka ROX kacha kwesị ekwesị maka ndị na-agba okirikiri fluorescence na-egosi na tebụl dị n'okpuru:

Tebụl 3-2: a na-enye ọnọdụ mmeghachi omume qPCR

Mara: Iji nweta nsonaazụ qPCR kacha mma, enwere ike iji gradient PCR kwalite ọnọdụ mmeghachi omume maka ndebiri dị iche iche yana primers dị iche iche.Ọnọdụ mmeghachi omume PCR dịgasị iche dabere na nyocha nke fluorescence, template, primer, wdg N'ime arụ ọrụ a kapịrị ọnụ, ọnọdụ mmeghachi omume kachasị mma kwesịrị ka emebere ya dabere na ọnọdụ dị iche iche nke fluorescence quantitative thermal cycler, ụdị template, nha nke iberibe mmasị, usoro ntọala nke mpekere gbasawanye na ọdịnaya GC na ogologo oge, gụnyere oge okpomọkụ, wdg.

Real Time PCR primer ụkpụrụ imewe

Na-aga n'ihu Primer na Reverse Primer

Maka Real Time PCR, imewe primer dị ezigbo mkpa.Primers metụtara nkọwapụta na arụmọrụ nke nkwalite PCR, enwere ike hazie ya na ntụnyere ụkpụrụ ndị a:

◆ Ogologo mbụ: 18-30bp.

◆ GC ọdịnaya: 40-60%.

◆ Tm uru: Akụrụngwa imewe nke mbụ, dị ka Primer 5, nwere ike inye uru Tm nke primer.Ụkpụrụ Tm nke ndị na-ebuli elu na nke mgbada kwesịrị ịdị nso dịka o kwere mee.A nwekwara ike iji usoro mgbako Tm: Tm = 4 °C (G + C) + 2 Celsius C (A + T).Mgbe ị na-arụ PCR, a na-ahọrọ okpomọkụ dị n'okpuru uru Tm primer nke 5 Celsius dị ka okpomọkụ na-ekpo ọkụ (mmụba kwekọrọ na okpomọkụ na-ekpo ọkụ nwere ike ịbawanye nkọwa nke mmeghachi omume PCR).

◆ Primers na PCR ngwaahịa:

◆ Design primer PCR amplification ogologo ngwaahịa bụ ọkachamma 100-150bp.

◆ Design primers na nke abụọ structural mpaghara nke template kwesịrị izere dị ka o kwere mee.

◆ Zenarị nhazi ntọala 2 ma ọ bụ karịa n'etiti nsọtụ 3 nke elu na ala ala.

◆ Primer 3′ ọdụ ọdụ enweghị ike ịnọ na 3 mgbakwunye G ma ọ bụ C ọzọ.

◆ The primers n'onwe ha enweghị ike inwe ihe mgbakwunye, ma ọ bụghị ya, a ga-emepụta usoro ntutu isi, na-emetụta mmụba PCR.

◆ Ekwesịrị kesaa ATCG n'otu n'otu dịka o kwere mee n'usoro nke mbụ, a ga-ezere ọdụ ọdụ 3′ dị ka T.

Ihe mgbakwunye1:Cebe DirectRT-qPCR Ngwakọta ihet mgbakwunye mkpọ

1.Cell Lysis Solution