Haufinyane tjena, ke fumane ntho e makatsang!Litsebi tse ngata tse tsoetseng pele tsa liteko tse mo potolohileng ha li tsebe le lintlha tsa motheo tsa tsebo ea liteko.

Ka mohlala, na u ka araba lipotso tse latelang?

Na ho na le phapang lipakeng tsa OD260 le A260?E 'ngoe le e 'ngoe e bolela'ng?
OD ke khutsufatso ea optical density (optical density), A ke khutsufatso ea absorbance (absorbance), likhopolo tse peli ha e le hantle li tšoana, "optical density" ke "absorbance", empa "optical density" e lumellana le litekanyetso tse ngata tsa naha le maemo a mangata.

Hangata re lekanya boleng ba OD ho 260nm ho bala mahloriso a nucleic acid, joale 1OD e emela eng?
Nucleic acid e na le tlhōrō e phahameng ea ho monya ka bolelele ba 260nm, e nang le DNA le RNA ka bobeli, hammoho le likhechana tsa nucleic acid (ena ke ntlha ea bohlokoa).
Boleng ba OD bo lekantsoeng ka bolelele ba 260 nm bo tlalehiloe e le OD260.Haeba sampole e hloekile, boleng ba OD260 bo ka bala bongata ba sampole ea nucleic acid.
1 OD260=50 μg/ml dsDNA (DNA e nang le likhoele tse peli)
=37 μg/ml ssDNA (DNA ea khoele e le 'ngoe)
=40 μg/mL RNA
=30 μg/ml dNTPs (oligonucleotides)
Na ho na le khokahano le phapang lipakeng tsa RT-PCR, Realtime-PCR le QPCR?
RT-PCR e khutsufetse bakeng sa Reverse Transcription PCR
Nako ea 'Nete PCR=qPCR, e khuts'oane bakeng sa Quantitative Real Time PCR
Leha ho le joalo Nako ea 'Nete PCR (ea sebele ea nako ea fluorescent quantitative PCR) le Reverse Transcription PCR (reverse transcription PCR) ka bobeli li bonahala li khutsufalitsoe e le RT-PCR.Empa kopano ea machaba ke: RT-PCR ka ho khetheha e bua ka reverse transcription PCR.

nt, bp, le kb tse sebelisoang hangata ho hlalosa bolelele ba DNA/RNA thutong ea baeloji ke life?
nt = nucleotide
bp = base pair base pair
kb = kilobase

Ha e le hantle, u ka re batho ba bangata ha ba tsotelle lintlha tsena tse nyenyane!E mong le e mong o etsa sena, mme ha ho motho ya tla o botsa hore na ke eng.Ua tseba hore sena ha se hlokahale, na?

Che, Che, Che, ho bohlokoa haholo ho tseba sena!ka lebaka la eng?
Hobane u batla ho beha sengoloa!Moena!Hore na u ikemiselitse ho fumana mangolo kapa ho phehella katleho ea lipatlisiso tsa mahlale, u tlameha ho itšetleha ka lingoliloeng ho bua!

Ho tlosoa ha Nucleic acid e lokela ho ba teko e bonolo le ea motheo ka ho fetisisa.Boleng ba nucleic acid extract ka ho toba bo khetholla liphello tsa liteko tse latelang.

Le hoja ke boletse hangata, ho ntse ho e-na le metsoalle e mengata e sa tsotelleng.Lekhetlong lena ke ile ka etsa qeto ea ho tsoa sehloohong seo!

Lintlha tse Nyenyane bakeng sa Phatlalatso ea Liteko tsa Nako ea 'Nete tsa Quantitative tsa PCR, tse bitsoang MIQE., ke sehlopha sa litataiso tsa liteko tsa fluorescence quantitative tse phatlalalitsoeng machabeng, tse sisinyang maemo a tlase bakeng sa tlhaiso-leseling ea liteko e hlokahalang bakeng sa ho hlahloba liteko tsa PCR tsa fluorescence le lingoliloeng.Ka maemo a liteko le mekhoa ea tlhahlobo e fanoeng ke mohlahlobi, bahlahlobisisi ba ka lekola hamolemo bonnete ba morero oa liteko oa mofuputsi.

Hoa hlokomeleha hore karolong ea nucleic acid extract, lintlha tse latelang tsa ho lemoha li 'nile tsa hlahisoa:

“E” e bontša tlhahisoleseding e lokelang ho fanoa ’me “D” e bontša tlhahisoleseding e lokelang ho fanoa ha ho hlokahala.

Foromo e rarahane haholo, ha e le hantle, ke batla ho bolela hore bohle ba hloka ho qala ho tloha

bohloeki (D), chai (D), botšepehi (E) le tumellano (E) ho hlahloba Nucleic acid likarolong tsena tse 'nè.

Ho ea ka mekhoa ea liteko, bua pele ka mekhoa ea tlhahlobo ea bohloeki le mahloriso.

Tekanyo ea OD ke mokhoa o ratoang le o bonolo ka ho fetisisa oa ho lemoha ho batho ba etsang liteko.Ha e le molao-motheo, nke ke ka kena lintlha tse qaqileng mona.Lilaboratori tse ngata joale li sebelisa li-ultra-micro spectrophotometer ho hlahloba ka ho toba lisampole tsa nucleic acid.Ha e ntse e bonts'a boleng ba ho monya, lenaneo le fana ka ho toba boleng ba mahloriso (nucleic acid, protheine le dae ea fluorescent) le likarohano tse amanang.Ha e le tlhahlobo ea boleng ba OD, Boloka setšoantšo sena 'me u tla phela hantle.

Universal OD lenane la tharollo ea boleng

Leha ho le joalo, ho na le litšitiso tse 'maloa tse lokelang ho hlahisoa ka thoko bakeng sa hau.

(Ka mor'a moo, kea tseba hore e tlameha ebe ke lōna ba bolokang chelete le ho ema ho fihlela le ba hloka!)

Hlokomela 1 Thepa

Boleng ba OD bo tla angoa ke lisebelisoa tse fapaneng.Ha feela OD260 e le ka har'a mefuta e itseng, boleng ba OD230 le OD280 bo na le moelelo.Ka mohlala, mefuta e mengata ea ho monya ea Eppendorf D30 e tloaelehileng ho 260nm ke 0~3A, 'me NanoDrop One ea Thermo e ho 260nm.Mofuta oa ho monya ke 0.5 ~ 62.5A.

Ntlha ea 2Dilution reagent

Boleng ba OD bo ka angoa ke ho hlapolloa ha li-reagents tse fapaneng.Mohlala, ho bala OD260/280 ea RNA e hloekisitsoeng ka pH7.5 10mM Trisbuffer e pakeng tsa 1.9-2.1, ha e ntse e le ka haretharollo e nang le metsi a sa nke lehlakorekaro-karolelano e tla ba tlase, mohlomong 1.8-2.0 feela, empa sena ha se bolele hore boleng ba RNA bo fetoha Phapang.

Ntlha ea 3Lintho tse setseng

Ho ba teng ha lintho tse setseng ho tla ama ho nepahala ha tekanyo ea mahloriso a nucleic acid, ka hona hoa hlokahala ho qoba masala a protheine, phenol, polysaccharide le polyphenol ho lisampole tsa asiti ea nucleic ka hohle kamoo ho ka khonehang.

Leha ho le joalo, ha e le hantle, ho ntšoa ka li-reagents tsa manyolo ke mokhoa oa khale.Likarolong tsa khoebo, phello ea ho ntšoa e ka finyelloa ka kholumo ea adsorption ea silica e kopantsoeng le centrifugation, ho qoba li-reagents tse chefo le tse kotsi tseo ho leng thata ho li tlosa, joalo-joalo.Foregene's nucleic acid extraction kit, ha e sebelise DNase/RNase le li-reagents tse chefo nakong eohle ea ts'ebetso, li potlakile ebile li bolokehile., le eaphello kemolemo(ka phoso o itse ke lefatla, empa kea tseba hore u batla ho tseba).

Mohlala 1: Kotulo ea ho ntšoa ha DNA ea Genomic le bohloeki

Foregene Soil DNA Isolation Kit (DE-05511) e tšoara lisampole tsa mobu ho tsoa mehloling e fapaneng, 'me palo le bohloeki ba DNA ea genomic e fumanoeng li bonts'oa lethathamong le latelang:
Mohlala 2: Kotulo ea ho ntšoa ha lithane tsa RNA le bohloeki

Animal Total RNA Isolation Kit (RE-03012) e sebelitse disampole tse fapaneng tsa dithishu, mme palo le bohlweki ba RNA bo fumanweng bo bontshwa tafoleng e ka tlase (bakeng sa dithishu tsa toeba):
Leha ho le joalo, u se ke oa nahana hore u se u qetile ka boleng ba OD.A na u na le lintlha tsa bohlokoa tseo ke u ngoletseng tsona ka pele?

Hlokomela

Limolek'hule tsa nucleic acid tse arohaneng le tsona li tla baloa ho monya.Ho nka hore u na le masala a DNA ea genomic ho RNA, boleng ba hau ba OD bo tla bonahala bo le holimo haholo, empa mahloriso a sebele a RNA a ke ke a tsejoa.Hore na RNA ea hau ke Ha ho hlake hore na ho na le ho senyeha, kahoo re ntse re hloka mokhoa o pharaletseng oa tlhahlobo ho fana ka kahlolo e nepahetseng haholoanyane, ke hore, tlhahlobo ea nucleic acid integrity e boletsoeng ho MIQE.