5×Neoscript Fast RT-qPCR Premix me-UNG

Helu popoki: HCB5142A

ʻO ka Neoscript Fast RT Premix-UNG (Probe qRT-PCR) he mea paʻa paʻa loa i hoʻokahi-pube probe-based mix kūpono no ka transcription hoʻohuli hoʻokahi a me ka PCR quantitative (qRT-PCR).Kākoʻo ia i ka hui mua ʻana o nā primers a me nā probes a noho paʻa ma hope o ka mālama ʻana i ka manawa lōʻihi i ka haʻahaʻa haʻahaʻa.Hiki ke hoʻohui pololei ʻia ka laʻana e hoʻāʻo ʻia i ka wā e hoʻohana ai, me ka ʻole o ka wehe ʻana o ka paipu.Hāʻawi kēia huahana i nā ʻāpana, e laʻa me ka hoʻomaka wela DNA polymerase, M-MLV, heat-labile uracil DNA glycosylase (TS-UNG), RNase Inhibitor, MgCl₂, nā dNTP (me ka dUTP ma kahi o ka dTTP), a me nā mea hoʻopaʻa.Me ka genetically modified rapid amplification reverse transcriptase a me DNA polymerase, hiki ke hoʻopau i ka PCR amplification i loko o 20-40 mau minuke.Hoʻohana kēia reagent i ka pale kūikawā no ka qPCR me nā enzyme hui ʻia o ka anti-inhibitory amplification enzyme a me UNG enzyme.No laila, hiki iā ia ke kiʻi i ka hoʻonui maikaʻi ʻana o nā genes i hoʻopaʻa ʻia a pale i ka hoʻonui hoʻopunipuni i hoʻokumu ʻia e ka PCR koena a me ka pollution aerosol.Ua kūpono kēia reagent me ka hapa nui o nā mea hana PCR fluorescence quantitative mai nā mea hana e like me Applied Biosystems, Eppendorf, Bio-Rad a me Roche.

ʻāpana

1.25×Neoscript Fast RTase/UNG Mix

2.5×Neoscript Fast RT Premix Buffer (dUTP)

Nā Kūlana Waihona

Pono e mālama ʻia nā ʻāpana āpau ma -20 ℃ no ka mālama lōʻihi a me 4 ℃ a hiki i 3 mahina.E ʻoluʻolu e hui pono ma hope o ka hoʻoheheʻe ʻana a me ka centrifuge ma mua o ka hoʻohana ʻana.Hōʻalo i ka hoʻoheheʻe pinepine ʻana.

qRT-PCR Reaction System Hoʻomākaukau

1) a.ʻO ka manaʻo hope loa o ka primer he 0.2μM.No nā hualoaʻa maikaʻi aʻe, hiki ke hoʻopaʻa ʻia ke kumu kumu i loko o ka laulā o 0.2-1μM.ʻO ka maʻamau, hiki ke hoʻopaʻa ʻia ka ʻimi noiʻi i loko o ka laulā o 0.1-0.3μM.

2) b. I ka hoʻohana ʻana i ke Kaʻina PCR wikiwiki, e hoʻonui i ka neʻe ʻana o nā primers a me nā probes hiki ke hopena i nā hopena amplification maikaʻi aʻe, a pono e hoʻonui ʻia ko lākou lakio e like me ia.

3) Loaʻa nā ʻano laʻana like ʻole i nā ʻano like ʻole a me nā ʻike o ka mea hoʻopaneʻe a me ka helu kope o ka gene i manaʻo ʻia.Pono e noʻonoʻo ʻia ka nui hāpana e ke kūlana maoli.E hana i ka dilution o ka hāpana me ka wai nuclease-free a i ʻole TE Buffer, inā pono.

Pane Cnā lāʻau lapaʻau

Ka Mana Mana

1.ʻIke hana: ʻike, kikoʻī a me ka hana hou ʻana o qPCR.

2.ʻAʻohe hana nuclease exogenous: ʻaʻohe exogenous endonuclease a me exonuclease pollution.

Nā memo

1.ʻAʻole emi ka nui o ka hoʻonui ʻana o ka DNA polymerase ma mua o 1kb/10s.Loaʻa i nā mea hana PCR ʻokoʻa ka wikiwiki hoʻomehana a me ka hoʻoluʻu, nā ʻano hoʻomalu wela a me ka conductivity thermal, a no laila pono ka hoʻonui ʻana i kāu kumu kumu/ʻimi noiʻi a me ke ʻano holo i hui pū ʻia me kāu mea hana PCR wikiwiki.

2.Hana kēia huahana i ka hoʻohana ākea, a kūpono ia no ka ʻike molecular high-sensitivity.Manaʻo ʻia ke ʻano hana PCR ʻekolu no nā primers me ka wela annealing haʻahaʻa a i ʻole no ka hoʻonui ʻana i nā ʻāpana lōʻihi ma luna o 200 bp.

3.No ka mea ʻokoʻa nā amplicons ʻokoʻa o ka hoʻohana ʻana o ka dUTP a me nā ʻano ʻokoʻa ʻē aʻe i ka UNG, pono e hoʻomaikaʻi ʻia nā reagents inā e emi ka ʻike ʻike i ka wā e hoʻohana ai i ka ʻōnaehana UNG.E ʻoluʻolu e kelepona mai iā mākou no ke kākoʻo ʻenehana inā pono.

4.I mea e pale aku ai i ka hoʻonui ʻia ʻana o nā huahana PCR carryover, pono ka wahi hoʻokolohua hoʻolaʻa a me ka pipette no ka hoʻonui.E hana me nā mīkina lima a hoʻololi pinepine a mai wehe i ka paipu PCR ma hope o ka hoʻonui ʻana.