Hoʻemi nui ʻo Kina High Sensitivity One-Step Probe Rt-Qpcr Kit V2
Ua ʻike mākou i ka mea hana.Ke lanakila nei i ka hapa nui i nā palapala hōʻoia koʻikoʻi o kāna mākeke no ka hoʻemi nui ʻana o Kina High Sensitivity One-Step Probe Rt-QpcrʻO Kit V2, ʻO ka maikaʻi ke ola o ka hale hana, ʻo ka nānā ʻana i ka noi o ka mea kūʻai aku ke kumu o ke ola ʻana o ka ʻoihana a me ka hoʻomohala ʻana, pili mākou i ka ʻoiaʻiʻo a me ka manaʻo maikaʻi o ka hana ʻana, e kali ana i kou hiki ʻana mai!
Ua ʻike mākou i ka mea hana.Ke lanakila nei i ka hapa nui o nā palapala hōʻoia koʻikoʻi o kāna mākeke noKina Taq DNA Polymerase, Qpcr, Hoʻohiki kā mākou hui: nā kumukūʻai kūpono, ka manawa hana pōkole a me ka lawelawe ma hope o ke kūʻai aku ʻoluʻolu, hoʻokipa pū mākou iā ʻoe e kipa i kā mākou hale hana i kēlā me kēia manawa āu e makemake ai.Makemake mākou i kēia manawa he ʻoihana ʻoluʻolu a lōʻihi hoʻi!!!
Nā wehewehe
Hoʻohana kēia kit i kahi ʻōnaehana lysis buffer kūʻokoʻa e hiki ke hoʻokuʻu koke i ka RNA mai nā laʻana cell moʻomeheu no nā hopena RT-qPCR, a laila e hoʻopau i ke kaʻina hoʻomaʻemaʻe RNA hoʻopau manawa a paʻakikī.Hiki ke loaʻa ka template RNA i loko o 7 mau minuke.Hiki i ka 5×Direct RT Mix a me ka 2×Direct qPCR Mix-SYBR reagents i hāʻawi ʻia e ka kit hiki ke loaʻa koke i nā hopena PCR quantitative maoli.
ʻO 5 × Direct RT Mix a me 2 × Direct qPCR Mix-SYBR he ikaika ka hoʻomanawanui inhibitor, a hiki ke hoʻohana ʻia ka lysate o nā laʻana e like me ke kumu hoʻohālike no RT-qPCR pololei.Aia kēia pahu i ka RNA high-affinity Foregene reverse transcriptase, a me Hot D-Taq DNA polymerase, dNTPs, MgCl2, pale pale, PCR optimizer a stabilizer.
Nā kikoʻī
200×20μl Rxns, 1000×20μl Rxns
Nā ʻāpana pahu
Mahele I | Hoʻopaʻa CL |
Foregene Protease Plus II | |
Hoʻopaʻa ST | |
Mahele II | DNA Holoi |
5× Direct RT Mix | |
2× Direct qPCR Mix-SYBR | |
50× ROX Mea Pepa Wai | |
RNase-Free ddH2O | |
Nā kuhikuhi |
Nā hiʻohiʻona & pono
■ Maʻalahi a maikaʻi : me ka ʻenehana Cell Direct RT, hiki ke loaʻa nā laʻana RNA i loko o 7 mau minuke.
■ He liʻiliʻi ka noi hāpana, hiki ke hoʻāʻo ʻia ma kahi haʻahaʻa he 10.
■ Kiʻekiʻe throughput: hiki iā ia ke ʻike koke i ka RNA i loko o nā cell moʻomeheu i 384, 96, 24, 12, 6-well plates.
■ Hiki i ka DNA Eraser ke wehe koke i nā genome i hoʻokuʻu ʻia, e hoʻemi nui i ka hopena ma nā hopena hoʻokolohua ma hope.
■ ʻOi aku ka maikaʻi o ka ʻōnaehana RT-PCR i ʻoi aku ka maikaʻi a me ka PCR i ʻoi aku ka maikaʻi, a ʻoi aku ke kūpaʻa ʻana i nā mea hoʻopaneʻe hopena RT-qPCR.
Ke noi pahu
Ka laulā o ka hoʻohana ʻana: nā cell moʻomeheu.
- Hoʻokuʻu ʻia ʻo RNA e ka laʻana lysis: pili wale i ka template RT-qPCR o kēia pahu.
- Hiki ke hoʻohana ʻia ke kit no nā kumu aʻe: ka nānā ʻana i ka hōʻike ʻana o ke ʻano, ka hōʻoia o ka hopena silencing gene-mediated gene siRNA, ka nānā ʻana i ka lāʻau, etc.
Diagram
Ka mālama ʻana a me ka mālama ʻana
Pono e mālama ʻia ka ʻāpana I o kēia pahu ma 4 ℃;Pono e mālama ʻia ka ʻāpana II ma -20 ℃.
Pono e mālama ʻia ka Foregene Protease Plus II ma 4 ℃, mai maloʻo ma -20 ℃.
Pono e mālama ʻia ka Reagent 2×Direct qPCR Mix-SYBR ma -20 ℃ i ka pōʻeleʻele;inā hoʻohana pinepineʻia, hiki ke mālamaʻia ma 4 ℃ no ka mālama manawa pōkole (hoʻohana i loko o nā lā 10). Uaʻike mākou i ka mea hana.ʻO ka lanakila ʻana i ka hapa nui o nā palapala hōʻoia koʻikoʻi o kāna mākeke no Big discounting China High Sensitivity One-Step Probe Rt-Qpcr Kit V2, Quality is factory' life, Focus on customer' demand is the source of company survival and development, Hoʻopili mākou i ka ʻoiaʻiʻo a me ka manaʻo maikaʻi o ka hana ʻana, ke nānā nei i kou hiki ʻana mai!
Hoʻemi nuiKina Taq DNA Polymerase, Qpcr, Hoʻohiki kā mākou hui: nā kumukūʻai kūpono, ka manawa hana pōkole a me ka lawelawe ma hope o ke kūʻai aku ʻoluʻolu, mahalo pū mākou iā ʻoe e kipa i kā mākou hale hana i kēlā me kēia manawa āu e makemake ai.Makemake mākou i kēia manawa he ʻoihana ʻoluʻolu a lōʻihi hoʻi!!!
QuickEasyTM Cell Direct RT-qPCR Kit -Taqman
Cat.No.DRT-01021/01022
No ke kelepona pololei RT-qPCR e hoʻohana ana i ≤ 1000,000 pūnaewele
Hoʻolauna huahana
Ke hoʻohana nei kēia huahana i kahi ʻōnaehana lysis buffer ʻokoʻa e hoʻokuʻu koke i ka RNA mai nā moʻomeheu cell samples no ka RT-qPCR reactions, e hoʻopau ana i ke kaʻina hana hoʻomaʻemaʻe RNA manawa a me ka hana, a me 7 mau minuke wale nō e kiʻi i ka template RNA i koi ʻia, me ka 5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman i hāʻawi ʻia e ka kit hiki ke loaʻa i nā hopena PCR manawa maoli.
5× Direct RT Mix a me 2× Direct qPCR Mix-Taqman loaʻa i ka hoʻomanawanui inhibitor ikaika a hiki ke hana i ka hoʻihoʻi maikaʻi a me ka hoʻonui kikoʻī me ka hoʻohana ʻana i ka lysate o ka hāpana e ana ʻia ma ke ʻano he template.Aia ka reagent i ka Foregene Reverse Transcriptase, Hot D-Taq DNA Polymerase, dNTPs, MgCl2, Reaction Buffer, PCR Optimizer a me Stabilizer, hiki ke hoʻohana ʻia me ka lysis buffer e ʻike wikiwiki a maʻalahi i nā laʻana, a loaʻa iā ia nā hiʻohiʻona o ka naʻau kiʻekiʻe, kikoʻī a me ke kūpaʻa.
Nā hiʻohiʻona huahana
ʻO ka ʻenehana Cell Direct RT maʻalahi a maʻalahi e lawe i nā minuke 7 e kiʻi i nā laʻana RNA.
He liʻiliʻi nā koi laʻana, a hiki ke hoʻohana ʻia ma kahi liʻiliʻi o 10 mau cell moʻomeheu no ka hoʻokolohua.
Kiʻekiʻe kiʻekiʻe no ka loaʻa wikiwiki ʻana o ka RNA o nā cell moʻomeheu e like me 384, 96, 24, 12, a me nā papa 6-well.
Hiki i ka DNA Eraser ke wehe koke i nā genome i hoʻokuʻu ʻia, e hōʻemi nui i ka hopena i nā hopena hoʻokolohua ma hope.
Hiki i nā ʻōnaehana RT a me qPCR i hoʻopaʻa ʻia i ʻelua mau ʻanuʻu RT-PCR me ka ʻoi aku ka maikaʻi o ka hoʻohuli ʻana i ka transcription, kikoʻī, a me ka ʻoi aku ka ikaika o ka RT-qPCR reaction inhibitor tolerance.
Ke noi pahu
Ka laulā o ka hoʻohana ʻana: Nā cell moʻomeheu.
ʻO ka laʻana lysis i unuhi ʻia i ka RNA: hoʻohana ʻia ma ke ʻano he ʻelua ʻanuʻu RT-qPCR template.
Hiki ke hoʻohana ʻia nā kits no kēia mau kumu: ka nānā ʻana i ka ʻōlelo hoʻoponopono gene, ka hoʻāʻo ʻana i nā allele, ka nānā ʻana i ka lāʻau, etc.
Nā palena pahu
Nā ʻāpana i hoʻonui ʻia ≤ 300 bp.
Hoʻohana ʻia nā kime i nā cell moʻomeheu hou.
Ka mana o ka maikaʻi o ka huahana
Wahi a ka FOREGENE's Total Quality Management System, ua ho'āʻo ikaikaʻia kēlā me kēia pūʻulu o nā pūʻulu pūʻulu Cell Direct RT-qPCR i nā manawa he nui e hōʻoia i ka hilinaʻi a me ka paʻa o ka maikaʻi o kēlā me kēia pūʻulu o nā pahu.
Kiko maʻiʻo
QuickEasyTM Cell Direct RT-qPCR Kit-Taqman | ||||
Nā ʻāpana pahu20μl qPCR Reaction System | DRT-01021 | DRT-01022 | Nānā | |
200 T | 1000 T | |||
Māhele I | Hoʻopaʻa CL | 4 ml | 20 ml |
Cell Lysis |
Foregene Protease Plus II | 80 μl | 400 μl | ||
Hoʻopaʻa ST | 400 μl | 1 ml × 2 | ||
Māhele II | DNA Holoi | 80 μl | 400 μl | |
5× Direct RT Mix * | 160 μl | 800 μl | RT | |
2× Direct qPCR Mix-Taqman * | 1 ml × 2 | 1.7 ml × 6 | qPCR | |
20×ROX Kuhikuhi Wailu | 40 μl | 200 μl | ||
RNase-Free ddH2O | 1.7 ml | 10 ml | ||
Papa kuhikuhi | 1 apana | 1 apana |
*:Cell Lysis, 5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman hiki ke kūʻai ʻokoʻa ʻia, hāʻawi ʻia nā kikoʻī ma Appendix 1 (PAGE 13).
Nā kūlana mālama
1. Nā Kūlana Hoʻouna
ʻO ke kaʻina holoʻokoʻa o ka halihali pahu hau hau haʻahaʻa, e hōʻoia i ka pahu i loko o ka mokuʻāina <4 °C.
2. Nā kūlana mālama
E mālama i ka ʻāpana I ma 4°C a me ka ʻāpana II ma -20°C.
Pono e mālama ʻia ka Foregene Protease Plus II ma 4°C, ʻaʻole i ka hau ma -20°C.
Mālama ʻia ka Reagent 2× Direct qPCR Mix-Taqman ma -20°C, a i ʻole ma 4°C no ka hoʻohana pōkole inā hoʻohana pinepine ʻia (i loko o 10 mau lā).
ʻIke ʻāpana kit
Buffer CL: Hāʻawi i ke kaiapuni i koi ʻia no nā hopena lysis cell.
Buffer ST: Hoʻopau i ka mea hana i loko o ka lysate e pale aku i nā hopena ma hope o RT.
DNA Eraser: DNA remover, ka hopena o ka wehe ʻana i ka genome ma nā hoʻokolohua ma hope.
5× Direct RT Mix: Loaʻa i ka pilina RNA kiʻekiʻe ʻo Foregene Reverse Transcriptase, RNase Inhibitor, dNTPs, stabilizers, enhancers, optimizers, a me ka hoʻohuli transcription primers no ka alignment maikaʻi loa (Random Primer, Oligo(dT)18Primer).
Foregene Protease Plus II: I loko o ka pōʻaiapili o ka lysis buffer, hoʻokuʻu ʻia nā cell e hoʻokuʻu i nā waikawa nucleic.
2× Direct qPCR Mix-Taqman: Aia kēia reagent i Hot D-Taq DNA Polymerase, dNTPs, MgCl2, hoʻopaʻa ʻana i ka hopena, PCR optimizer, a me ka stabilizer.
20× ROX Reference Dye: Hoʻohana maʻamau i nā mea hoʻonui PCR Real Time o ABI, Stratagene a me nā ʻoihana ʻē aʻe, hoʻohana ʻia ia e hoʻoponopono i ka ʻokoʻa ma waena o nā paipu PCR a me nā paipu i hoʻokumu ʻia e nā hewa dosing PCR.ʻOkoʻa ka 20× ROX Reference Dye i koi ʻia no nā mea kani like ʻole, a hiki i ka mea hoʻohana ke hoʻohui iā ia e like me ka manaʻo o ka mea kani.
RNase-Free ddH2O: RNase-free sterilized ultra clean water no two-step RT-qPCR reactions.
Nā mea e mālama ai:(E heluhelu pono i nā kaʻina hana ma mua o ka hoʻohana ʻana i ka pahu)
E noʻonoʻo i ke ʻano hana o ka hoʻokolohua e pale aku i ka hoʻohaumia ʻana ma waena o nā laʻana.
E noʻonoʻo i ka maʻemaʻe o ke kaiapuni hoʻokolohua a me nā mea hana e pale aku i ka haumia RNase a me ka hōʻino ʻana o RNA.
E lawe i nā laʻana cell hou a i mālama pono ʻia a mai hoʻohana pinepine i nā laʻana pūnaewele i hoʻoheheʻe ʻia.
5× Direct qPCR Mix, 2× Direct qPCR Mix-Taqman pono e pale i ka hoʻokuʻu hou ʻana, inā ʻaʻole e pili ia i ka unuhi ʻana a me ka pono PCR.
Prepaʻai ʻaimamuahana
E heluhelu pono i nā ʻōlelo kuhikuhi ma mua o ka hoʻohana ʻana i kēia pahu.He maʻalahi, maʻalahi, a wikiwiki hoʻi ka Cell Direct RT-qPCR Kit, a hāʻawi nā kuhikuhi i ka ʻike piha e pili ana i ka pahu holoʻokoʻa a pehea e hoʻohana pono ai.E ʻoluʻolu e hoʻomākaukau i nā mea hoʻokolohua pono a me nā mea hana ma mua o ka hoʻohana ʻana.
Nā mea hoʻokolohua a me nā mea hana
◆ Nā pūnaewele moʻomeheu.
◆ 1.5 ml a i ʻole 2 ml, RNase-/DNase-Free centrifuge paipu, RNase-/DNase-Free tip, 0.2 ml sterile qPCR paipu.
◆ mīkini qPCR, pipette, centrifuge papa (≥13,400×g) (ma muli o nā pono hoʻokolohua), etc.
Palekana
◆ No ka noiʻi ʻepekema wale nō kēia huahana, mai hoʻohana ʻoe no ka lāʻau lapaʻau, lāʻau lapaʻau, meaʻai a me nā kumu hoʻonaninani.
◆ I ka wā e hoʻohana ai i nā kemika, e hoʻokomo i nā lole lab kūpono, nā mīkina lima, nā aniani pale, a pēlā aku.
Hanaalakai
Hiki ke kūʻai kaʻawale ʻia nā ʻōnaehana Cell Lysis, ʻōnaehana RT, a me qPCR reaction solution solution, no nā kikoʻī ma Appendix 1 (PAGE 13).
alakaʻi hana
A: Laʻana RNA hoʻokuʻu
1. Hoʻomaʻamaʻa mua ʻia nā kelepona: holoi i ka pā moʻomeheu cell me ka PBS anu, a laila e hoʻoheheʻe i nā cell (10-10).6), 106 ma mua o ka nui o nā pūnaewele, ua ʻōlelo ʻia ʻo Foregene the Cell a RNA Isolation Kit ka Total (DE-03111) a i ʻole Animal Total RNA Isolation Kit (DE-03011) no ka unuhi ʻana a me ka hoʻomaʻemaʻe RNA.
1.1.Nā pūnaewele pili (24- well plate ma ke ʻano he laʻana)
1.1.1.E hoʻoholo i ka helu o nā pūnaewele i kēlā me kēia pūnāwai, e hoʻoholo i ka helu o nā pūnaewele he 1 × 105, a hoʻohana i ka pipette e wehe i ka mea moʻomeheu mai ka pā moʻomeheu.
1.1.2.E hoʻohui i 200 μl o 1 × PBS i hoʻomaloʻo mua ʻia i kēlā me kēia pūnāwai.Mai paipu pinepine a wehe i ka PBS mai nā pūnāwai.Huli i ka pā a wehe i ka PBS e like me ka hiki.E hoʻomau i ka ʻanuʻu 2.
1.1.3.Ua hoʻohui ʻia kahi kīʻaha moʻomeheu kelepona ʻokoʻa a i ʻole ka papa helu kuhikuhi 1-1 i loko o kahi kīʻaha moʻomeheu cell i hoʻohui ʻia ʻo 1 × PBS no ka holoi kelepona.
Papa 1-1: PBS no nā helu like ʻole o nā cell
ʻAno papa moʻomeheu | Ka helu o nā pūnaewele / pūnāwai | 1 × PBS/ pūnāwai |
6-maikaʻi | 1× 106 | 1000 μl |
12-well | 2× 105 | 400 μl |
24-maikaʻi | 105 | 200 μl |
96-maikaʻi | 104 | 50 μl |
384-maikai | 5× 103 | 25 μl |
Nānā:No ka hoʻopaʻa paʻa ʻana i nā cell pili,ʻalo ʻia ka nui o ka nalowale o ka cell i ka wā e holoi ai.
1.2.Nā pūnaewele hoʻokuʻu ʻia a i ʻole nā pūnaewele pili i moʻomeheu ʻia i loko o nā papa porous ʻole
1.2.1.ʻO nā pūnaewele pili i moʻomeheu ʻia i loko o nā pā puna wai ʻole (hoʻomaka nā mea hoʻokuʻu ʻia mai ka ʻanuʻu aʻe 1.2.2), e hōʻiliʻili a hoʻokaʻawale i nā cell e like me ke ʻano o ka hōʻiliʻili cell maʻamau, a waiho iā lākou i loko o ka pā moʻomeheu a i ʻole centrifuge tube;inā hoʻohana ʻia ka trypsinization, koi ʻia ka centrifugation e hōʻiliʻili i nā cell a e hoʻoneʻe i ke koena trypsin, hoʻohui ʻia nā cell PBS resuspended i loko o nā pūnaewele pākahi e hoʻopuehu i nā cell.
1.2.2.Ma hope o ka helu ʻana o nā cell i helu ʻia, hoʻokaʻawale ʻia nā cell 1 × 105 hoʻokahi i nā paipu centrifuge, e hōʻiliʻili i nā pūnaewele ma ka centrifugation ma 1000 × g no 10 min.
1.2.3.E hoʻohui i 200 μl PBS i ka paipu centrifuge, mai paipu pinepine, a e hoʻomoʻi pololei i ka PBS.e hoʻomau i ka ʻanuʻu 2. (Inā paʻakikī ke hoʻoheheʻe ʻia a hoʻomaha hou ʻia nā cell, hiki ke hana ʻia 1000×g centrifuged 10 min ma hope o ka hoʻolei ʻana i ka supernatant, hoʻomau ka pellet cell i ka pae 2)
2.Cell lysis: Wehe Buffer CL, kona wela equilibrated i ka lumi wela, DNA Eraser a me Foregene Protease Plus II, e like me keia papa 1-2 hoomakaukau lysis nenoaiu: (Lysis solution is ready for use).
Papa 1-2: mahele hoʻomākaukau ʻōnaehana (Note: i ka hoʻomākaukau ʻana i ka hau)
ʻāpana (Ka Hui Pū ʻIa Lysis Master Mix) | 6-pūnāwai pā | 12-puna puna | 24-puna puna | 96-puna puna | 384-puna puna |
1000 μl / pūnāwai | 400 μl / pūnāwai | 200 μl / punawai | 50 μl / pūnāwai | 25 μl / pūnāwai | |
Hoʻopaʻa CL | 960μl | 384μl | 192μl | 48μl | 24μl |
DNA Holoi | 20μl | 8μl | 4μl | 1μl | 0.5 μl |
Foregene Protease Plus II | 20μl | 8μl | 4μl | 1 μl | 0.5 μl |
3.( 24 –well plate e like me ka laʻana) Pipette 200 μl o ka cell lysis master hui i loko o kēlā me kēia pūnāwai, E puhi pinepine i 5-10 manawa, Incubate ma ka lumi wela (20-25 ℃) no 5 min.
Nānā:I mea e pale aku ai i ka hoʻokumu ʻana o nā ʻōhū, e ʻoluʻolu i ka wā i hoʻoponopono ʻia ai ka nui pipette pipette i 200μl a i ʻole.Hiki ke ʻike ʻia ke ao ma hope o ka lysis, he mea maʻamau.
4. (24 –well plate e like me ka laʻana) ua hoʻohui ʻia i loko o ka wai 20 μl Buffer ST (nā ʻōnaehana lysis ʻokoʻa Buffer ST i hoʻohui ʻia i kahi nui i hōʻike ʻia ma ka Papa 1-3), hoʻopiʻi hou ʻia i ka pipetting 5-10 mau manawa, ma ka lumi wela (20-25 ℃) ua incubated no 2 min.
Nānā:Hoʻokuʻu ʻia ka piko pipette ma lalo o ka ʻili, e hōʻoia i ka hoʻohui ʻia o ka lysate,no ka pale ʻana i ka hoʻokumu ʻana o nā ʻūhū, e ʻoluʻolu i ka wā i hoʻoponopono ʻia ai ka pipetting scale pipette i 200μl a i ʻole.
Papa 1-3:Hoʻohui i ka Buffer ST
Hoʻopaʻa ST | 6- papa punawai | 12- papa punawai | 24- punawai papa | 96- punawai papa | 384- papa punawai |
100 μl / pūnāwai | 40 μl / pūnāwai | 20 μl / pūnāwai | 5 μl / pūnāwai | 2.5 μl / pūnāwai |
5. Hoʻohana ʻia ka lysate no nā hoʻokolohua RT-qPCR ma hope.Inā ʻaʻole hiki ke hoʻokō ʻia nā hoʻokolohua ma hope i ka manawa, e ʻoluʻolu e mālama iā ia ma ka hau no ka ʻoi aku o 2hr, a e mālama i -20 ℃ a i ʻole -80 ℃ (ʻaʻole ʻoi aku ma mua o ʻekolu mahina).
B: Hoʻomākaukau ʻōnaehana RT
1. E lawe i waho 5 × Direct RT Mix a kau ma luna o kahi hau hau, e hoʻoheheʻe maoli, a hui mālie no ka hoʻohana ʻana ma hope;e lawe i ka RNase-Free ddH2O a hoʻoheheʻe ʻia a kau ma luna o ka ʻauʻau hau no ka hoʻohana ʻana ma hope.E hoʻomākaukau i ka ʻōnaehana pane ma ka hau e like me ka Papa 2-1 ma lalo nei.
Papa 2-1: Ka hoʻomākaukau ʻana i ka ʻōnaehana pane RT
ʻO ka ʻōnaehana RT e hoʻohui i nā ʻike | Me ka nui | Hoʻoholo hope | |
5 × Direct RT Mix | 4μl | 8 μl | 1 × |
Nā Lysates pūnaewele (RNA template) | 4 μl | 8 μl | Hoʻohui i ka hoʻoponopono ākea (10 -40%) |
RNase-Free ddH2O | 12 μl | 24 μl | - |
Huina Volume | 20 μl | 40 μl | - |
2. Ma hope o ka pau ʻana o ka hoʻokumu ʻana o ka ʻōnaehana, hui ʻia a centrifuged pōkole i ka papa ma lalo nei 2 -2 mau kūlana hopena RT.
Papa 2-2: Hoʻonohonoho kūlana RT Reaction
ʻanuʻu | Mahana | manawa | maʻiʻo |
1 | 42 °C | 15-30 min | ka hoʻohui ʻana o ka cDNA |
2 | 95 °C | 5 min | ʻAʻole hoʻohana ʻia ka reverse transcriptase |
3 | 4 °C | N/A |
3. Ma hope o ka pau ʻana o ka hopena, ua kau pololei ʻia ka huahana hopena ma luna o ka hau no qPCR, e ʻoluʻolu e kau i ka mālama lōʻihi -20 ℃ a i ʻole -80 ℃.
'Ōlelo Aʻo: Ma muli o ka hoʻohana ʻana i ka laʻana i hoʻomaʻemaʻe ʻole ʻia, hiki ke ʻike ʻia nā precipitates keʻokeʻo ma ka huahana transcription hope.He mea maʻamau kēia.E hoʻonanea koke i ka supernatant no nā hoʻokolohua ma hope.
Hoʻohui ʻia ka hopena hopena RT i ka ʻōnaehana hopena ʻo Step Real Time PCR, pono e hoʻohui i nā helu mai ka 10-30% o ka ʻōnaehana pane.
C: qPCR hoʻomākaukau ʻōnaehana ʻōnaehana
1. Ka nui kūpono o ka B i hoʻomākaukau ʻia ma ka ʻanuʻu cDNA template e like me ka papa 3-1 aʻe e hoʻomākaukau ai i kahi ʻōnaehana pane.
'Ōlelo Aʻo: ʻO ka nui o ka cDNA template no 10-30% o ka ʻōnaehana qPCR.No ka laʻana, i loko o kahi ʻōnaehana qPCR 20μl, hoʻohui i 2-6 μl o ka lysis buffer, akā ʻaʻole ʻoi aku ma mua o 6 μl.
2. The optimization maikaʻi qPCR kūlana (Annealing wela,etc.) no ka qPCR pane (The reaction conditions given at Table 3-2).
'Ōlelo Aʻo: E ho'āʻo e hoʻohana i nā kūlana kūpono no nā pane qPCR no ka loaʻa ʻana o nā hopena maikaʻi aʻe.
Papa 3-1: Ka hoʻomākaukau ʻana i ka ʻōnaehana hana PCR
ʻO ka ʻōnaehana RT e hoʻohui i nā ʻike | Me ka nui | Hoʻoholo hope |
2× Direct qPCR Mix-Taqman | 10 μl | 1× |
Kuhi mua (10μM) | 0.4 μl | 50-900 nM 1* |
Pumi Hoʻohuli (10μM) | 0.4 μl | 50-900 nM 1* |
ʻImi (10μM) | 0.2 μl | 200nM |
cDNA template (loaʻa ma ka ʻanuʻu B) | 4 μl | 10-30% |
RNase-Free ddH2O | — | |
20×ROX Mea Waikane 3* | — | — |
Huina Volume | 20 μl |
1*: Hiki ke hoʻololi ʻia ka manaʻo kumu mua ma ka laulā o 50-900 nM i ka wā maikaʻi ʻole ka hana ʻana o ka hopena.
'Ōlelo Aʻo: Hiki ke hoʻololi ʻia ka ʻōnaehana qPCR e like me nā pono hoʻokolohua a me ke kumu hoʻohālike fluorescence cycler.No ka qPCR i kahi 50μl ka ʻōnaehana, hoʻoponopono i ka dosage reagent e like me ka 20μl ʻōnaehana.
Mīkini PCR manawa maoli | ʻO ROX Reference Dye ka manaʻo hope loa |
ABI PRISM7000/7300/7700/7900HT/Step One, etc. | 1×(e laʻa. 20 μl ʻōnaehana,hoʻohui i 1 μl 20×ROX Hōʻike Pelo) |
ABI 7500/7500 wikiwiki a me StratageneMx3000P/Mx3005P/Mx4000, etc. | 0.5×(e laʻa. 20 μl ʻōnaehana, hoʻohui 0.5 μl 20×ROX ReferenceDye) |
2*: E koho i ka manaʻo hope kūpono o ROX Reference Dye e like me ka fluorescence quantitative thermal cycler.Hōʻike ʻia nā koina ʻokoʻa ROX Reference Dye no nā mea hoʻokele nui fluorescence maʻamau i ka papa ma lalo.
Papa 3-2: Hāʻawi ʻia nā kūlana hoʻonā qPCR
ʻElua-ʻanuʻu | Mahana | Manawa | Nā pōʻaiapuni | Maʻiʻo |
1 | 95 ℃ | 3 min | 1 | Predenaturation |
2 | 95 ℃ | 5-10 sec | 40 | denaturation template |
3 | 60-65 ℃ | 20-30 sec | Hoʻopili / Hoʻonui |
'Ōlelo Aʻo: No ka loaʻa ʻana o ka hopena qPCR maikaʻi loa, hiki ke hoʻohana ʻia ka gradient PCR no ka hoʻopaʻa ʻana i nā kūlana pane no nā mamana like ʻole a me nā primers like ʻole.ʻOkoʻa nā kūlana hopena PCR ma muli o ka mea ananaly fluorescence, template, primer, etc. I ka hana kikoʻī, pono e hoʻolālā ʻia nā kūlana hopena maikaʻi loa e like me nā kūlana kikoʻī o ka fluorescence quantitative thermal cycler, ʻano template, ka nui o ka ʻāpana o ka hoihoi, ke kaʻina kumu o ka ʻāpana amplified a me GC maʻiʻo a me ka lōʻihi o nā primers, me ka annealing wela, ka manawa pane, etc.
Nā loina hoʻolālā mua PCR manawa maoli
Kuhikuhi i mua a me ke kumu mua
No Real Time PCR, he mea nui ka hoʻolālā mua.Pili nā kumu mua i ka kikoʻī a me ka pono o ka hoʻonui ʻana i ka PCR, a hiki ke hoʻolālā ʻia me ka pili ʻana i nā kumu aʻe:
◆ Ka lōʻihi mua: 18-30bp.
◆ Maʻiʻo GC: 40-60%.
◆ Waiwai Tm: Hiki i nā polokalamu hoʻolālā kumu, e like me Primer 5, ke hāʻawi i ka waiwai Tm o ke kumu.Pono e kokoke loa na koina Tm o na kumu mua a me lalo.Hiki ke hoʻohana ʻia ke kumu helu Tm: Tm = 4 °C (G + C) + 2 °C (A + T).Ke hana ʻia ka PCR, koho ʻia ka wela ma lalo o ka waiwai Tm kumu mua o 5 °C ma ke ʻano he wela annealing (hiki ke hoʻonui ʻia ka hoʻonui ʻana o ka mahana annealing i ka kikoʻī o ka hopena PCR).
◆ Nā mea mua a me nā huahana PCR:
◆ ʻOi aku ka maikaʻi o ka 100-150bp ka lōʻihi o ka hoʻonui ʻana o ka huahana PCR.
◆ Pono e hōʻalo ʻia nā mea hoʻolālā hoʻolālā ma ka ʻāpana kūkulu lua o ka template i ka hiki.
◆ Hōʻalo i ka hoʻokumu ʻana o 2 a ʻoi aʻe paha kumu hoʻohui ma waena o nā 3′ welau o nā kumu mua a me lalo.
◆ ʻAʻole hiki ke loaʻa ke kumu kumu kumu 3′ me 3 G a i ʻole C.
◆ ʻAʻole hiki i nā primers ke loaʻa nā hale hoʻohui, inā ʻaʻole e hana ʻia kahi ʻano lauoho lauoho, e pili ana i ka PCR amplification.
◆ Pono e puunaue like 'ia ka ATCG e like me ka hiki ma ke ka'ina mua, a e 'alo 'ia ke kumu kumu 3′ e like me T.
Pākuʻi1:Cell DirectRT-qPCR ʻāpana pahut pūʻolo hoʻohui
1.Cell Lysis Solution
| |||
Nā ʻāpana pahu (24-well lysis system / well) | DRT-01011-A1 | DRT-01011-A2 | |
100 T | 500 T | ||
MāheleI | Hoʻopaʻa CL | 20 ml | 100 ml |
Foregene Protease Plus II | 400 μl | 1 ml × 2 | |
Hoʻopaʻa ST | 1 ml × 2 | 10 ml | |
MāheleII | DNA Holoi | 400 μl | 1 ml × 2 |
| |
Nā ʻāpana pahu (20 μl ʻōnaehana pane) | DRT-01011-B1 |
200 T | |
5× Direct RT Mix | 800 μl |
RNase-Free ddH2O | 1.7 ml × 2 |
3.qPCR Hui
| ||
Nā ʻāpana pahu (20 μl ʻōnaehana pane) | DRT-01021-C1 | DRT-01021-C2 |
200 T | 1000 T | |
2× Direct qPCR Mix-Taqman | 1 ml × 2 | 1.7 ml × 6 |
20× ROX Peʻa Waikane | 40 μl | 200 μl |
RNase-Free ddH2O | 1.7 ml | 10 ml |
Ka Makana o ka Honua
Foregene Co., Ltd
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E-mail :info@foregene.com
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