Mperutse, navumbuye ikintu gitangaje!Abashakashatsi benshi bateye imbere bamuzengurutse ntibazi nubumenyi bwibanze bwubushakashatsi.

Kurugero, ushobora gusubiza ibibazo bikurikira?

Haba hari itandukaniro hagati ya OD260 na A260?Buri wese asobanura iki?
OD ni impfunyapfunyo yubucucike bwa optique (ubwinshi bwa optique), A ni impfunyapfunyo yo kwinjiza (absorbance), ibyo bitekerezo byombi mubyukuri ni bimwe, "ubucucike bwa optique" ni "kwinjiza", ariko "ubwinshi bwa optique" bujyanye nibipimo byinshi byigihugu kandi birasanzwe.

Mubisanzwe dupima agaciro ka OD kuri 260nm kugirango tubare aside nucleic yibanze, none 1OD igereranya iki?
Acide Nucleic ifite igipimo ntarengwa cyo kwinjirira ku burebure bwa 260nm, ikubiyemo ADN na RNA, hamwe n'ibice bya acide nucleic yacitsemo ibice (iyi ni yo ngingo y'ingenzi).
Agaciro ka OD gapimye ku burebure bwa 260 nm yanditswe nka OD260.Niba icyitegererezo ari cyiza, agaciro ka OD260 karashobora kubara ubunini bwa acide nucleic.
1 OD260 = 50 μg / ml dsDNA (ADN ikubye kabiri)
= 37 μg / ml ssDNA (ADN imwe imwe)
= 40 μg / ml RNA
= 30 μg / ml dNTPs (oligonucleotide)
Haba hari isano n'itandukaniro hagati ya RT-PCR, Igihe-PCR na QPCR?
RT-PCR ni ngufi kuri Reverse Transcription PCR
Igihe nyacyo PCR = qPCR, ngufi kuri Quantitative Real Time PCR
Nubwo Igihe nyacyo PCR (real-time fluorescent quantitative PCR) na Reverse Transcription PCR (reverse transcription PCR) byombi bisa nkaho bigufi nka RT-PCR.Ariko amasezerano mpuzamahanga ni: RT-PCR yerekeza cyane cyane kwandukura PCR.

Ni ubuhe buryo bukunze gukoreshwa nt, bp, na kb mu gusobanura uburebure bwa ADN / RNA muri biologiya?
nt = nucleotide
bp = ishingiro ryibanze
kb = kilobase

Birumvikana, wavuga ko abantu benshi batitaye kuri utuntu duto duto!Umuntu wese arabikora, kandi ntamuntu uzakubaza icyo aricyo.Uzi ko ibi bidakenewe, sibyo?

Oya, Oya, Oya, birakenewe cyane kubimenya!kubera iki?
Kuberako ushaka kohereza ingingo!Muvandimwe!Waba ugamije kurangiza cyangwa gukurikirana ibyagezweho mubushakashatsi bwa siyansi, ugomba kwishingikiriza ku ngingo zo kuvuga!

Gukuramo aside nucleique bigomba kuba ubushakashatsi bworoshye kandi bwibanze.Ubwiza bwo gukuramo aside nucleique bugena neza ibisubizo byubushakashatsi bwakurikiyeho.

Nubwo nabivuze inshuro nyinshi, haracyari inshuti nyinshi zitabyitayeho.Iki gihe nahisemo kuva mu ngingo!

Amakuru ntarengwa yo gutangaza umubare wuzuye-Igihe PCR Ubushakashatsi, bwitwa MIQE, ni urutonde rwibipimo ngenderwaho bya fluorescence byatangijwe ku rwego mpuzamahanga, bitanga ibipimo ntarengwa byamakuru yubushakashatsi akenewe mugusuzuma ibipimo bya fluorescence PCR no gutangaza ingingo.Binyuze mubihe byubushakashatsi nuburyo bwo gusesengura butangwa nuwabigerageje, abasesengura barashobora gusuzuma neza agaciro ka gahunda yubushakashatsi.

Birashobora kugaragara ko mugice cyo gukuramo aside nucleic, hashyizweho ibintu bikurikira byo gutahura,

“E” yerekana amakuru agomba gutangwa naho “D” yerekana amakuru agomba gutangwa nibiba ngombwa.

Ifishi iragoye cyane, mubyukuri, ndashaka kuvuga ko abantu bose bakeneye guhera

ubuziranenge (D), umusaruro (D), ubunyangamugayo (E) no guhuzagurika (E) gusuzuma aside Nucleic muri izi ngingo enye.

Ukurikije akamenyero ko kugerageza, banza uvuge uburyo bwo gusuzuma isuku no kwibanda.

Ibipimo bya OD nuburyo bukunzwe kandi bworoshye bwo kumenya kubashakashatsi.Kubijyanye nihame, ntabwo nzajya muburyo burambuye hano.Laboratoire nyinshi ubu zikoresha ultra-micro spectrophotometero kugirango isesengure mu buryo butaziguye urugero rwa acide nucleic.Mugihe herekana agaciro ko kwinjiza, porogaramu itanga mu buryo butaziguye agaciro kokwibanda (aside nucleic, proteine ​​na fluorescent irangi) hamwe nibipimo bifitanye isano.Kubijyanye no gusesengura agaciro ka OD, Bika iyi shusho kandi uzaba mwiza.

Urutonde rusange rwa OD agaciro

Ariko, hariho caveats nkeya zigomba kuzanwa ukwawe kubwawe.

(Nyuma ya byose, nzi ko ugomba kuba ariwe uzigama ugategereza kugeza ubakeneye!)

Icyitonderwa 1 Ibikoresho

Agaciro ka OD kazagerwaho nibikoresho bitandukanye.Igihe cyose OD260 iri murwego runaka, indangagaciro za OD230 na OD280 zirasobanutse.Kurugero, urwego rwo kwinjiza Eppendorf D30 isanzwe kuri 260nm ni 0 ~ 3A, naho NanoDrop Imwe muri Thermo ni 260nm.Urwego rwo kwinjiza rwa 0.5 ~ 62.5A.

Icyitonderwa 2Dilution reagent

Agaciro ka OD karashobora guhindurwa no kugabanuka kwa reagent zitandukanye.Kurugero, OD260 / 280 gusoma RNA yatunganijwe muri pH7.5 10mM Trisbuffer iri hagati ya 1.9-2.1, mugihe muriigisubizo kidafite aho kibogamiyeigipimo kizaba gito, wenda 1.8-2.0 gusa, ariko ibi ntibisobanura ko ubwiza bwa RNA buhindura Itandukaniro.

Icyitonderwa 3Ibintu bisigaye

Kubaho kw'ibisigara bizagira ingaruka ku gupima ibipimo bya acide nucleique, bityo rero birakenewe ko twirinda poroteyine, fenol, polysaccharide hamwe na polifenol ibisigisigi bya aside nucleique bishoboka.

Ariko, mubyukuri, gukuramo hamwe ningingo ngengabuzima ni uburyo bwa kera.Mu bikoresho byubucuruzi, ingaruka zo gukuramo zishobora kugerwaho hifashishijwe inkingi ya silika ishingiye kuri adsorption hamwe na centrifugation, ukirinda ubumara bwangiza kandi bwangiza ibintu bigoye kuvanaho, nibindi. Ikibazo, nkaForegene yo gukuramo aside nucleic, ntabwo ikoresha DNase / RNase hamwe nubumara bwa organic reagent mugikorwa cyose, byihuse kandi bifite umutekano, naIngaruka nibyiza(kubwimpanuka yavuze ko byari uruhara, ariko nzi ko ushaka kubimenya).

Urugero rwa 1: Gukuramo ADN genomic umusaruro nubuziranenge

Ubutaka bwa Foregene ADN Isolation Kit (DE-05511) ivura ingero zubutaka buturuka ahantu hatandukanye, kandi ingano nubuziranenge bwa ADN genomic yabonetse byerekanwe kumeza ikurikira:
Urugero rwa 2: Tissue RNA ikuramo umusaruro nubuziranenge

Amatungo Yuzuye ya RNA Yigunga (RE-03012) yatunganije ingero zinyuranye, kandi ingano nubuziranenge bwa RNA yabonetse byerekanwe kumeza hepfo (kubice byimbeba):
Ariko, ntutekereze ko urangije agaciro ka OD.Waba ufite icyo wita ku ngingo z'ingenzi nagushushanyije imbere?

Menyesha

Molekile ya nucleic acitsemo ibice nayo izabarwa mukwinjira.Dufashe ko ufite ibisigisigi bya ADN bisigara muri RNA, agaciro ka OD kazagaragara nkaho kari hejuru cyane, ariko kwibanda kwa RNA ntigushobora kumenyekana.Niba RNA yawe itarasobanutse neza niba hariho gutesha agaciro, turacyakeneye rero uburyo bwuzuye bwo gusuzuma kugirango dutange imyanzuro ihamye, ni ukuvuga isuzuma rya acide nucleic acide yavuzwe muri MIQE.