Munguva pfupi yapfuura, ndakawana chimwe chinhu chinoshamisa!Nyanzvi zhinji dzepamusoro dzekuyedza dzakamutenderedza havatomboziva mamwe mapoinzi eruzivo rwekutanga.

Somuenzaniso, unogona kupindura mibvunzo inotevera here?

Pane musiyano pakati peOD260 neA260?Chimwe nechimwe chinorevei?
OD ndiyo chidimbu che optical density (optical density), A ndiyo chidimbu cheabsorbance (absorbance), pfungwa mbiri idzi dzakafanana, “optical density” ndi“absorbance”, asi “optical density” inofambirana nezviyero zvakawanda zvenyika uye zvakamisikidzwa.

Kazhinji isu tinoyera kukosha kweOD pa260nm kuverenga iyo nucleic acid concentration, saka 1OD inomiririra chii?
Nucleic acid ine yakanyanya kunyura peak pawavelength ye260nm, iyo ine zvese DNA neRNA, pamwe neakatsemuka nucleic acid zvidimbu (iyi ndiyo yakakosha poindi).
Kukosha kweOD kuyerwa pawavelength ye260 nm yakanyorwa seOD260.Kana iyo sampuli yakachena, iyo OD260 kukosha inogona kuverenga kuwanda kweiyo nucleic acid sampu.
1 OD260=50 μg/ml dsDNA (DNA ine tambo mbiri)
=37 μg/ml ssDNA (DNA ine tambo imwe chete)
=40 μg/ml RNA
=30 μg/ml dNTPs (oligonucleotides)
Pane here kubatana uye musiyano pakati peRT-PCR, Realtime-PCR neQPCR?
RT-PCR ipfupi yeReverse Transcription PCR
Real Time PCR=qPCR, ipfupi yeQuantitative Real Time PCR
Kunyangwe Real Time PCR (chaiyo-nguva fluorescent quantitative PCR) uye Reverse Transcription PCR (reverse transcription PCR) zvese zvinoita sekudimburirwa seRT-PCR.Asi gungano repasi rese nderekuti: RT-PCR inonyatso inoreva kudzoreredza chinyorwa PCR.

Ndeapi anonyanya kushandiswa nt, bp, uye kb kutsanangura kureba kweDNA/RNA mubhayoroji?
nt = nucleotide
bp = base pair base pair
kb = kilobase

Chokwadi, ungati vanhu vakawanda havana basa nezvinhu zviduku izvi!Munhu wese anoita izvi, uye hapana achakubvunza kuti chii.Iwe unoziva izvi hazvifanirwe, handiti?

Kwete, Kwete, Kwete, zvakakosha kuziva izvi!nekuda kwei?
Nekuti unoda kutumira chinyorwa!Hanzvadzi konama!Kunyangwe iwe uchivavarira kupedza chikoro kana kutsvaga kubudirira kwesainzi, unofanirwa kuvimba nezvinyorwa kuti utaure!

Nucleic acid kutorwa kunofanirwa kuve kuri nyore uye kwakanyanya kuyedza.Hunhu hweiyo nucleic acid kubviswa zvakanangana nemhedzisiro yezviedzo zvinotevera.

Kunyange ndakazvitaura kakawanda, kuchine shamwari dzakawanda dzisina basa nazvo.Panguva ino ndakasarudza kubuda munyaya yacho!

Ruzivo Rwakaderera rweKushambadza Kwehuwandu Chaiyo-Nguva PCR Kuedza, inonzi MIQE., seti ye fluorescence quantitative experiment guidelines yakatangwa pasi rose, iyo inopa mipimo mishoma yeruzivo rwekuyedza runodiwa pakuongorora fluorescence quantitative PCR zviedzo uye kutsikisa zvinyorwa.Kuburikidza nemamiriro ekuedza uye nzira dzekuongorora dzakapihwa nemuongorori, vaongorori vanogona kuongorora zviri nani chokwadi chechirongwa chemuongorori.

Zvinogona kuoneka kuti muchikamu cheiyo nucleic acid kudhirowa, zvinotevera zvinoonekwa zvinhu zvakatsanangurwa,

“E” inoratidza ruzivo runofanira kupihwa uye “D” inoratidza ruzivo runofanira kupihwa kana zvichidikanwa.

Iyo fomu yakaoma kwazvo, chokwadi, ndinoda kutaura kuti munhu wese anofanira kutanga kubva

kuchena (D), goho (D), kuvimbika (E) uye kusachinja-chinja (E) kuongorora Nucleic acids mune izvi zvinhu zvina.

Zvinoenderana nemaitiro ekuyedza, tanga taura nezve nzira dzekuongorora dzekuchena uye kuisa pfungwa.

OD kuyerwa ndiyo inofarira uye iri nyore nzira yekuona yevanoedza.Kana iri musimboti, ini handisi kuzopinda mune zvakadzama pano.Marabhoritari mazhinji zvino anoshandisa Ultra-micro spectrophotometers kuti aongorore zvakananga nucleic acid samples.Ichiri kuratidza kukosha kwekunyudza, chirongwa chinopa zvakananga kukosha kweiyo (nucleic acid, protein uye fluorescent dhayi) uye zvine hukama.Kana iri yekuongorora kweiyo OD kukosha, Chengetedza mufananidzo uyu uye uchave wakanaka.

Universal OD kukosha mhinduro runyorwa

Nekudaro, pane mashoma mashoma anofanirwa kuburitswa akaparadzana iwe.

(Mushure mezvose, ndinoziva kuti munofanira kunge muri imi munochengeta uye kumirira kusvikira mavakuda!)

Cherechedza 1 Equipment

Kukosha kweOD kunokanganiswa nemidziyo yakasiyana.Chero bedzi iyo OD260 iri mukati mehumwe danho, kukosha kweOD230 neOD280 zvine zvazvinoreva.Semuenzaniso, iyo absorbance range yeyakajairika Eppendorf D30 pa260nm ndeye 0 ~ 3A, uye NanoDrop Imwe yeThermo iri pa260nm.Iyo yekunyudza huwandu hwe0.5 ~ 62.5A.

Note 2Dilution reagent

Kukosha kweOD kunogona kukanganiswa nekuderedzwa kwemareagents akasiyana.Semuenzaniso, iyo OD260/280 kuverenga kweRNA yakacheneswa mupH7.5 10mM Trisbuffer iri pakati pe1.9-2.1, ichiri mukatineutral aqueous solutioniyo reshiyo ichava yakaderera, pamwe chete 1.8-2.0, asi izvi hazvirevi kuti kunaka kweRNA kunoshandura Kusiyana.

Cherechedza 3Zvisaririra zvinhu

Kuvepo kwezvinhu zvakasara kuchakanganisa kurongeka kweiyo nucleic acid yekumisikidza kuyerwa, saka zvinodikanwa kudzivirira mapuroteni, phenol, polysaccharide uye polyphenol masara mune nucleic acid samples zvakanyanya sezvinobvira.

Zvisinei, chaizvoizvo, kubudiswa ne organic reagents inzira yekare.Mumakiti ekutengeserana, maitiro ekubvisa anogona kuwanikwa kuburikidza ne silica-based adsorption column yakasanganiswa ne centrifugation, kudzivisa chepfu uye inokuvadza organic reagents iyo yakaoma kubvisa, nezvimwewo Dambudziko, rakadai seForegene's nucleic acid extraction kit, haishandise DNase/RNase uye zvine muchetura organic reagents mukati mekushanda, nekukurumidza uye kwakachengeteka., uyemhedzisiro ndiyokugona(netsaona akati yaive mhanza, asi ndinoziva kuti unoda kuziva).

Muenzaniso 1: Genomic DNA kutora goho uye kuchena

Foregene Soil DNA Isolation Kit (DE-05511) inobata masampuli evhu kubva kwakasiyana siyana, uye huwandu uye kuchena kwegenomic DNA yakawanikwa inoratidzwa mune inotevera tafura:
Muenzaniso 2: Tissue RNA kuburitsa goho uye kuchena

Mhuka Yese RNA Isolation Kit (RE-03012) yakagadziridzwa akasiyana matishu samples, uye huwandu nekuchena kweRNA yakawanikwa inoratidzwa patafura iri pazasi (yematishu embeva):
Nekudaro, usafunge kuti waitwa neiyo OD kukosha.Iwe une chero chekuchengetedza mapoinzi akakosha andakakukwevera iwe pamberi?

Cherechedzai

Yakatsemuka nucleic acid mamorekuru anozoverengerwawo mukutora.Tichifunga kuti une genomic DNA zvakasara muRNA, kukosha kweOD yako ichaita seyakanyanya kukwirira, asi iyo chaiyo yeRNA haigone kuzivikanwa.Kana RNA yako iriHazvina kujeka kana pane kudzikisirwa, saka tichiri kuda nzira yekuongorora yakazara kuti tipe mutongo wakanyanya, kureva, nucleic acid kuvimbika evaluation inotaurwa muMIQE.