Tsis ntev los no, kuv nrhiav tau ib yam dab tsi amazing!Ntau tus kws tshaj lij kev sim siab nyob ib puag ncig nws tsis txawm paub qee cov ntsiab lus kev sim paub yooj yim heev.

Piv txwv li, koj tuaj yeem teb cov lus nug hauv qab no?

Puas muaj qhov sib txawv ntawm OD260 thiab A260?Txhua tus txhais li cas?
OD yog cov tsiaj ntawv luv ntawm qhov muag ceev (kho qhov muag tsis zoo), aquical densatory "yog" kho qhov muag "yog nyob rau hauv txoj kab nrog feem ntau cov qauv hauv tebchaws thiab ntau dua.

Feem ntau peb ntsuas tus nqi OD ntawm 260nm los xam cov nucleic acid concentration, yog li 1OD sawv cev li cas?
Nucleic acid muaj qhov siab tshaj plaws nqus ntawm lub wavelength ntawm 260nm, uas muaj ob qho tib si DNA thiab RNA, nrog rau cov nucleic acid fragmented fragmented (qhov no yog lub ntsiab lus tseem ceeb).
Tus nqi OD ntsuas ntawm lub wavelength ntawm 260 nm tau sau tseg ua OD260.Yog tias tus qauv yog ntshiab, tus nqi OD260 tuaj yeem xam qhov concentration ntawm nucleic acid qauv.
1 OD260 = 50 μg / ml dsDNA (ob-stranded DNA)
= 37 μg/ml ssDNA (ib leeg-stranded DNA)
= 40 μg / ml RNA
= 30 μg / ml dNTPs (oligonucleotides)
Puas muaj kev sib txuas thiab qhov sib txawv ntawm RT-PCR, Realtime-PCR thiab QPCR?
RT-PCR luv luv rau Reverse Transcription PCR
Real Time PCR = qPCR, luv rau Quantitative Real Time PCR
Txawm hais tias Real Time PCR (real-time fluorescent quantitative PCR) thiab Reverse Transcription PCR (reverse transcription PCR) ob qho tib si zoo li raug hu ua RT-PCR.Tab sis lub rooj sib tham thoob ntiaj teb yog: RT-PCR tshwj xeeb yog hais txog kev thim rov qab PCR.

Dab tsi yog qhov feem ntau siv nt, bp, thiab kb los piav txog qhov ntev ntawm DNA / RNA hauv biology?
nt = nucleotide
bp = base Pair Pair
kb = kilobase

Tau kawg, koj yuav hais tias ntau tus neeg tsis quav ntsej txog cov ntsiab lus me me no!Txhua tus ua li no, thiab tsis muaj leej twg yuav nug koj tias nws yog dab tsi.Koj paub tias qhov no tsis tsim nyog, txoj cai?

Tsis yog, Tsis yog, Tsis yog, nws yog qhov tsim nyog yuav tsum paub qhov no!vim qhov twg?
Vim koj xav tshaj tawm ib tsab xov xwm!Kwv tij!Txawm hais tias koj tab tom npaj rau kev kawm tiav lossis nrhiav kev tshawb fawb txog kev ua tiav, koj yuav tsum tso siab rau cov lus hais!

Nucleic acid extraction yuav tsum yog qhov yooj yim tshaj plaws thiab feem ntau sim.Qhov zoo ntawm nucleic acid extraction ncaj qha txiav txim siab cov txiaj ntsig ntawm kev sim tom ntej.

Txawm hais tias kuv tau hais ntau zaus los tseem muaj ntau tus phooj ywg uas tsis quav ntsej.Lub sijhawm no kuv txiav txim siab tawm ntawm tsab xov xwm!

Cov ntaub ntawv yam tsawg kawg nkaus rau kev tshaj tawm ntawm Quantitative Real-Time PCR Experiments, hu ua MIQE, yog cov txheej txheem ntawm fluorescence quantitative thwmsim cov txheej txheem tau tsim tawm thoob ntiaj teb, uas tawm tswv yim yam tsawg kawg nkaus rau kev sim cov ntaub ntawv tsim nyog rau kev ntsuam xyuas fluorescence quantitative PCR thwmsim thiab luam tawm cov ntawv.Los ntawm cov kev sim thiab cov txheej txheem kev soj ntsuam muab los ntawm tus kws sim, cov neeg soj ntsuam tuaj yeem ntsuas qhov siv tau zoo ntawm tus kws tshawb fawb txoj kev sim.

Nws tuaj yeem pom tau tias nyob rau hauv ntu ntawm nucleic acid extraction, cov khoom tshawb nrhiav hauv qab no tau raug npaj,

"E" qhia cov ntaub ntawv uas yuav tsum tau muab thiab "D" qhia cov ntaub ntawv uas yuav tsum tau muab yog tias tsim nyog.

Daim ntawv yog qhov nyuaj heev, qhov tseeb, kuv xav hais tias txhua tus yuav tsum pib los ntawm

purity (D), yield (D), kev ncaj ncees (E) thiab sib xws (E) los ntsuas Nucleic acids hauv plaub yam no.

Raws li kev sim cwj pwm, thawj zaug tham txog cov kev ntsuam xyuas ntawm purity thiab concentration.

Kev ntsuas OD yog txoj kev nyiam thiab nrhiav tau yooj yim tshaj plaws rau cov neeg sim.Raws li txoj cai, kuv yuav tsis mus rau hauv cov ntsiab lus ntawm no.Ntau lub chaw soj nstuam tam sim no siv ultra-micro spectrophotometers ncaj qha txhawm rau txheeb xyuas cov qauv nucleic acid.Thaum pom tus nqi absorbance, qhov kev pab cuam ncaj qha muab tus nqi concentration (nucleic acid, protein thiab fluorescent dye) thiab lwm yam piv.Raws li kev txheeb xyuas tus nqi OD, Txuag daim duab no thiab koj yuav zoo.

Universal OD tus nqi daws cov npe

Txawm li cas los xij, muaj qee qhov lus ceeb toom uas yuav tsum tau coj tawm nyias rau koj.

(Tom qab tag nrho, kuv paub tias koj yuav tsum yog cov uas txuag thiab tos kom txog thaum koj xav tau lawv!)

Nco tseg 1 Khoom siv

Tus nqi OD yuav raug cuam tshuam los ntawm cov khoom sib txawv.Tsuav yog OD260 nyob rau hauv ib qho chaw, qhov tseem ceeb ntawm OD230 thiab OD280 yog qhov tseem ceeb.Piv txwv li, qhov absorbance ntau yam ntawm Eppendorf D30 ntawm 260nm yog 0 ~ 3A, thiab NanoDrop Ib qho ntawm Thermo yog ntawm 260nm.Lub absorbance ntau ntawm 0.5 ~ 62.5A.

Nco tseg 2Dilution reagent

Tus nqi OD tuaj yeem cuam tshuam los ntawm dilution ntawm cov reagents sib txawv.Piv txwv li, OD260/280 nyeem ntawm purified RNA hauv pH7.5 10 mM Tristsis yog nyob nruab nrab ntawm 1.9-2.1, thaum nyob rau hauvnruab nrab aqueous tovqhov piv yuav qis dua, tej zaum tsuas yog 1.8-2.0, tab sis qhov no tsis txhais tau tias qhov zoo ntawm RNA hloov qhov txawv.

Nco tseg 3Cov khoom seem

Lub hav zoov ntawm cov khoom seem yuav cuam tshuam qhov tseeb ntawm nucleic acid concentration ntsuas, yog li nws yuav tsum tsis txhob muaj cov protein, phenol, polysaccharide thiab polyphenol residues hauv cov qauv nucleic acid kom ntau li ntau tau.

Txawm li cas los xij, qhov tseeb, kev rho tawm nrog cov organic reagents yog txoj kev qub.Nyob rau hauv cov khoom siv coj mus muag, cov nyhuv extraction tuaj yeem ua tiav los ntawm silica-based adsorption kem ua ke nrog centrifugation, tsis txhob muaj tshuaj lom thiab teeb meem organic reagents uas nyuaj rau tshem tawm, thiab lwm yam. Cov teeb meem, xws liForegene's nucleic acid extraction kit, tsis siv DNase / RNase thiab tshuaj lom cov organic reagents thoob plaws hauv kev ua haujlwm, ceev thiab nyab xeeb, thiab coveffect yogzoo(tam sim no hais tias nws yog khaus, tab sis kuv paub koj xav paub).

Piv txwv 1: Genomic DNA extraction yield thiab purity

Foregene Av DNA Isolation Kit (DE-05511) kho cov qauv av los ntawm ntau qhov chaw, thiab tus nqi thiab purity ntawm genomic DNA tau txais yog qhia hauv cov lus hauv qab no:
Piv txwv 2: Tissue RNA extraction yield thiab purity

Tsiaj Tag Nrho RNA Isolation Kit (RE-03012) tau ua tiav ntau yam qauv ntaub so ntswg, thiab tus nqi thiab purity ntawm RNA tau muaj nyob rau hauv cov lus hauv qab no (rau cov ntaub so ntswg nas):
Txawm li cas los xij, tsis txhob xav tias koj ua tiav nrog tus nqi OD.Koj puas muaj kev saib xyuas cov ntsiab lus tseem ceeb uas kuv kos rau koj hauv ntej?

Daim ntawv ceeb toom

Fragmented nucleic acid molecules kuj yuav xam nyob rau hauv lub absorbance.Piv txwv tias koj muaj genomic DNA residues hauv RNA, koj tus nqi OD yuav tshwm sim siab heev, tab sis qhov tseeb ntawm RNA tsis tuaj yeem txiav txim siab.Txawm hais tias koj tus RNA tsis paub meej tias puas muaj kev puas tsuaj, yog li peb tseem xav tau txoj kev ntsuam xyuas dav dav kom muab kev txiav txim kom raug ntau dua, uas yog, kev ntsuam xyuas kev ncaj ncees nucleic acid tau hais hauv MIQE.