I kēia mau lā, ua ʻike au i kahi mea kupaianaha!ʻAʻole ʻike ka nui o nā poʻe loea hoʻokolohua kiʻekiʻe a puni iā ​​​​ia i kekahi mau wahi ʻike kumu hoʻokolohua.

Eia kekahi laʻana, hiki iā ʻoe ke pane i nā nīnau ma lalo nei?

Aia kekahi ʻokoʻa ma waena o OD260 a me A260?He aha ke ʻano o kēlā me kēia?
ʻO OD ka pōkole o ka optical density (optical density), A ʻo ia ka pōkole o ka absorbance (absorbance), ʻo nā manaʻo ʻelua he like maoli nō, ʻo "optical density" ka "absorbance", akā ʻo ka "optical density" e pili ana me ka hapa nui o nā kūlana aupuni a ʻoi aku ka maʻamau.

He mea maʻamau mākou e ana i ka waiwai OD ma 260nm no ka helu ʻana i ka nui o ka waika nucleic, no laila he aha ka 1OD?
ʻO ka waika Nucleic kahi kiʻekiʻe absorption peak ma kahi hawewe o 260nm, aia i loko o ka DNA a me ka RNA, a me nā ʻāpana nucleic acid i ʻokiʻoki ʻia (ʻo ia ke kī nui).
Ua hoʻopaʻa ʻia ka waiwai OD i ana ʻia ma kahi hawewe nalu o 260 nm e like me OD260.Inā maʻemaʻe ka hāpana, hiki i ka waiwai OD260 ke helu i ka neʻe ʻana o ka hāpana nucleic acid.
1 OD260=50 μg/ml dsDNA (DNA pae ʻelua)
= 37 μg/ml ssDNA (DNA kaula hoʻokahi)
=40 μg/ml RNA
=30 μg/ml dNTPs (oligonucleotides)
Aia kekahi pilina a ʻokoʻa ma waena o RT-PCR, Realtime-PCR a me QPCR?
He pōkole ʻo RT-PCR no Reverse Transcription PCR
PCR Manawa Maoli=qPCR, pōkole no Quantitative Real Time PCR
ʻOiai ʻo Real Time PCR (real-time fluorescent quantitative PCR) a me Reverse Transcription PCR (reverse transcription PCR) me he mea lā ua hoʻopau ʻia ʻo RT-PCR.Akā ʻo ka hui honua: RT-PCR e pili ana i ka PCR transcription hoʻohuli.

He aha nā nt, bp, a me kb i hoʻohana mau ʻia no ka wehewehe ʻana i ka lōʻihi o DNA/RNA i loko o ka biology?
nt = nucleotide
bp = hui kumu kumu
kb = kilobase

ʻOiaʻiʻo, e ʻōlelo paha ʻoe he nui ka poʻe i mālama ʻole i kēia mau kikoʻī liʻiliʻi!Hana nā kānaka a pau i kēia, ʻaʻohe mea e nīnau iā ʻoe he aha ia.ʻIke ʻoe he mea pono ʻole kēia, ʻeā?

ʻAʻole, ʻAʻole, ʻAʻole, pono loa e ʻike i kēia!no ke aha?
No ka mea makemake ʻoe e kau i kahi ʻatikala!Kaikuaʻana, kaikaina!Inā ʻoe e ʻimi nei i ka puka ʻana a i ʻole ka ʻimi ʻana i nā holomua noiʻi ʻepekema, pono ʻoe e hilinaʻi i nā ʻatikala e ʻōlelo ai!

ʻO ka unuhi ʻana i ka waika nucleic ka hoʻokolohua maʻalahi loa.Hoʻoholo pololei ka maikaʻi o ka ʻili o ka nucleic acid i nā hopena o nā hoʻokolohua ma hope.

ʻOiai ua ʻōlelo pinepine au, nui nā hoa aloha ʻole.I kēia manawa ua hoʻoholo wau e haʻalele i ka ʻatikala!

ʻIke liʻiliʻi loa no ka hoʻolaha ʻana i nā mea hoʻokolohua PCR manawa maoli, i kapa ʻia ʻo MIQE, he pūʻulu o nā alakaʻi hoʻokolohua fluorescence quantitative hoʻokolohua i hoʻokuʻu ʻia ma ke ao holoʻokoʻa, e hāpai ana i nā kūlana haʻahaʻa no ka ʻike hoʻokolohua e pono ai no ka loiloi ʻana i nā hoʻokolohua PCR quantitative fluorescence a me ka hoʻopuka ʻana i nā ʻatikala.Ma o nā kūlana hoʻokolohua a me nā ʻano loiloi i hāʻawi ʻia e ka mea hoʻokolohua, hiki i nā mea loiloi ke loiloi maikaʻi i ka pono o ka hoʻolālā hoʻokolohua a ka mea noiʻi.

Hiki ke ʻike ʻia ma ka ʻāpana o ka ʻili o ka nucleic acid, ua manaʻo ʻia nā mea ʻike ma lalo nei,

Hōʻike ʻo "E" i ka ʻike pono e hāʻawi ʻia a ʻo "D" hōʻike i ka ʻike e pono e hāʻawi ʻia inā pono.

He paʻakikī loa ke ʻano, ʻoiaʻiʻo, makemake wau e ʻōlelo pono e hoʻomaka nā mea a pau

ka maʻemaʻe (D), ka hua (D), ka pono (E) a me ke kūlike (E) e loiloi i nā waikawa Nucleic i kēia mau ʻano ʻehā.

Wahi a nā hana hoʻokolohua, e kamaʻilio mua e pili ana i nā ʻano loiloi o ka maʻemaʻe a me ka noʻonoʻo.

ʻO ke ana ʻo OD ke ala ʻike punahele a maʻalahi loa no nā mea hoʻokolohua.No ke kumu, ʻaʻole wau e hele i nā kikoʻī ma aneʻi.Nui nā hale hana i kēia manawa e hoʻohana i nā spectrophotometers ultra-micro no ka hoʻopili pololei ʻana i nā mea hoʻohālike nucleic acid.ʻOiai e hōʻike ana i ka waiwai absorbance, hāʻawi pololei ka papahana i ka waiwai hoʻopaʻa (nucleic acid, protein and fluorescent dye) a me nā ratio pili.E pili ana i ka loiloi o ka waiwai OD, E mālama i kēia kiʻi a maikaʻi ʻoe.

Papa helu hoʻonā waiwai OD

Eia nō naʻe, aia kekahi mau ʻōlelo hōʻoia e pono e lawe ʻia i waho no ʻoe.

(Ma hope o nā mea a pau, ʻike wau ʻo ʻoe ka mea nāna e mālama a kali a pono ʻoe iā lākou!)

Nānā 1 Mea lako

E hoʻopili ʻia ka waiwai OD e nā lako like ʻole.ʻOiai ka lōʻihi o ka OD260 i loko o kahi kikoʻī, pono nā waiwai o OD230 a me OD280.No ka laʻana, ʻo ka laulā absorbance o ka Eppendorf D30 maʻamau ma 260nm ʻo 0 ~ 3A, a ʻo ka NanoDrop One of Thermo aia ma 260nm.ʻO ka laulā absorbance o 0.5 ~ 62.5A.

Nānā 2Mea hoʻoheheʻe hoʻoheheʻe

Hiki ke hoʻopili ʻia ka waiwai OD e ka dilution o nā reagents ʻokoʻa.No ka laʻana, ʻo ka heluhelu OD260/280 o ka RNA i hoʻomaʻemaʻe ʻia i ka pH7.5 10mM ʻO TrisAia ka buffer ma waena o 1.9-2.1, ʻoiai i lokohoʻoheheʻe wai kū ʻolee emi ka lakio, malia paha he 1.8-2.0 wale nō, akā ʻaʻole kēia manaʻo e hoʻololi ka maikaʻi o ka RNA i ka ʻokoʻa.

Nānā 3Nā mea i koe

ʻO ka noho ʻana o nā mea koena e hoʻopilikia i ka pololei o ke ana ʻana o ka nucleic acid, no laila pono e pale aku i ka protein, phenol, polysaccharide a me nā koena polyphenol i nā mea hoʻohālike nucleic acid e like me ka hiki.

Eia naʻe, ʻoiaʻiʻo, ʻo ka unuhi ʻana me nā reagents organik kahi ala kahiko.I loko o nā pahu pāʻoihana, hiki ke loaʻa ka hopena unuhi ma o ke kolamu adsorption e pili ana i ka silica i hui pū ʻia me ka centrifugation, e pale ana i nā mea ʻona a me nā mea ʻino i paʻakikī e wehe, etc. ʻO ka pilikia, e like meʻAʻole hoʻohana ʻo Foregene's nucleic acid extraction kit i ka DNase/RNase a me nā mea ʻawaʻawa ʻawaʻawa i ka wā o ka hana, wikiwiki a palekana., a me kaʻo ka hopenamaikaʻi loa(Ua ʻōlelo ʻia he ʻōhule, akā ʻike wau makemake ʻoe e ʻike).

Ka Laʻana 1: Genomic DNA extraction yield and cleanly

Mālama ʻo Foregene Soil DNA Isolation Kit (DE-05511) i nā laʻana lepo mai nā kumu like ʻole, a ʻo ka nui a me ka maʻemaʻe o ka DNA genomic i loaʻa e hōʻike ʻia ma ka papa aʻe:
Ka Laʻana 2: ʻO ka hoʻohua a me ka maʻemaʻe o ka ʻili RNA

Ua hoʻoponopono ʻia ka Animal Total RNA Isolation Kit (RE-03012) i nā laʻana kikoo like ʻole, a hōʻike ʻia ka nui a me ka maʻemaʻe o ka RNA i loaʻa ma ka papa ma lalo nei (no ka ʻiole).
Eia naʻe, mai noʻonoʻo ua pau ʻoe i ka waiwai OD.Loaʻa iā ʻoe kahi mālama i nā mea nui aʻu i huki ai no ʻoe ma mua?

Hoolaha

E helu pū ʻia nā molekole nucleic acid i hoʻopaʻa ʻia.Ke manaʻo nei he koena DNA genomic kāu i loko o ka RNA, e ʻike ʻia kāu waiwai OD he kiʻekiʻe loa, akā ʻaʻole hiki ke hoʻoholo i ka ʻike maoli o ka RNA.Inā paha kāu RNA ʻAʻole maopopo inā he degradation, no laila pono mākou i kahi ʻano loiloi piha e hāʻawi i ka hoʻoholo pololei, ʻo ia hoʻi, ka loiloi nucleic acid integrity i ʻōlelo ʻia ma MIQE.