Mea Hana Kina no ka 2× Concentrated Premix no ka laʻana ʻaʻole i hoʻomaʻemaʻe ʻia i ka manawa maoli Quantitative PCR Direct Multiplex Probe Qpcr Mix Plus U
Hoʻomau ka hui i ka manaʻo kaʻina hana "ka hoʻokele ʻepekema, kūlana kiʻekiʻe a me ka pono primacy, mea kūʻai kiʻekiʻe no Kina Manufacturer no 2 × Concentrated Premix for Unpurified Sample Real-Time Quantitative PCR Direct Multiplex Probe Qpcr Mix Plus U, Hoʻolaʻa kā mākou ʻoihana i ka hāʻawi ʻana i nā mea kūʻai aku me nā huahana kiʻekiʻe a paʻa i ke kumukūʻai koʻikoʻi.
Hoʻomau ka hui i ka manaʻo kaʻina hana "ka hoʻokele ʻepekema, kūlana kiʻekiʻe a me ka maikaʻi primacy, mea kūʻai kiʻekiʻe noKina Taq DNA Polymerase a me Qpcr, Nui ka ikaika o kā mākou hui a loaʻa iā ia kahi ʻōnaehana kūʻai kūpaʻa a maikaʻi loa.Makemake mākou e hiki iā mākou ke hoʻokumu i nā pilina ʻoihana maikaʻi me nā mea kūʻai aku a pau mai ka home a i waho ma ke kumu o nā pōmaikaʻi like.
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 loaʻa ʻana o ka lōʻihi o ka huahana PCR amplification primer 100-150bp.
- Pono e pale ʻia nā kumu mua o ka hoʻolālā ʻana ma ka ʻāpana kūkulu lua o ka template i ka mea hiki.
- Hōʻalo i ka hoʻokumu ʻia ʻana o nā kumu hoʻohui ʻelua a ʻoi aʻe paha ma waena o nā welau 3′ o nā kumu mua a me lalo.
- ʻAʻole hiki ke loaʻa ke kumu kumu kumu 3′ me 3 mau G a i ʻole C.
- ʻAʻole hiki i nā primers ke loaʻa nā hale hoʻohui, inā ʻaʻole e hoʻokumu ʻia kahi ʻano lauoho lauoho, e pili ana i ka hoʻonui PCR.
- Pono e puunaue like 'ia ka ATCG e like me ka hiki ma ke ka'ina mua, a e pale 'ia ke kumu 3' e like me T.
Pākuʻi1:Cell DirectRT-qPCR ʻāpana pahut pūʻolo hoʻohui
1.Cell Lysis Solution
ʻO ka hoʻonā cell Lysis | |||
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 |
RT Mix | |
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 |
Huina qPCR | ||
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 |
Hoʻomau ka hui i ka manaʻo kaʻina hana "ka hoʻokele ʻepekema, kūlana kiʻekiʻe a me ka pono primacy, mea kūʻai kiʻekiʻe no Kina Manufacturer no 2 × Concentrated Premix for Unpurified Sample Real-Time Quantitative PCR Direct Multiplex Probe Qpcr Mix Plus U, Hoʻolaʻa kā mākou ʻoihana i ka hāʻawi ʻana i nā mea kūʻai aku me nā huahana kiʻekiʻe a paʻa i ke kumukūʻai koʻikoʻi.
Kina Manufacturer noKina Taq DNA Polymerase a me Qpcr, Nui ka ikaika o kā mākou hui a loaʻa iā ia kahi ʻōnaehana kūʻai kūpaʻa a maikaʻi loa.Makemake mākou e hiki iā mākou ke hoʻokumu i nā pilina ʻoihana maikaʻi me nā mea kūʻai aku a pau mai ka home a i waho ma ke kumu o nā pōmaikaʻi like.
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 loaʻa ʻana o ka lōʻihi o ka huahana PCR amplification primer 100-150bp.
- Pono e pale ʻia nā kumu mua o ka hoʻolālā ʻana ma ka ʻāpana kūkulu lua o ka template i ka mea hiki.
- Hōʻalo i ka hoʻokumu ʻia ʻana o nā kumu hoʻohui ʻelua a ʻoi aʻe paha ma waena o nā welau 3′ o nā kumu mua a me lalo.
- ʻAʻole hiki ke loaʻa ke kumu kumu kumu 3′ me 3 mau G a i ʻole C.
- ʻAʻole hiki i nā primers ke loaʻa nā hale hoʻohui, inā ʻaʻole e hoʻokumu ʻia kahi ʻano lauoho lauoho, e pili ana i ka hoʻonui PCR.
- Pono e puunaue like 'ia ka ATCG e like me ka hiki ma ke ka'ina mua, a e pale 'ia ke kumu 3' e like me T.
Pākuʻi1:Cell DirectRT-qPCR ʻāpana pahut pūʻolo hoʻohui
1.Cell Lysis Solution
ʻO ka hoʻonā cell Lysis | |||
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 |
RT Mix | |
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 |
Huina qPCR | ||
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 |
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