Posachedwapa, ndapeza chinthu chodabwitsa!Akatswiri ambiri oyesera apamwamba omwe amamuzungulira sadziwa ngakhale mfundo zina zofunika kwambiri zoyesera.

Mwachitsanzo, kodi mungayankhe mafunso otsatirawa?

Kodi pali kusiyana pakati pa OD260 ndi A260?Kodi chilichonse chimatanthauza chiyani?
OD ndi chidule cha kachulukidwe kuwala (optical kachulukidwe), A ndi chidule cha absorbance (absorbance), mfundo ziwiri kwenikweni zofanana, "optical kachulukidwe" ndi "absorbance", koma "optical kachulukidwe" amagwirizana ndi mfundo zambiri dziko ndi yokhazikika.

Nthawi zambiri timayesa mtengo wa OD pa 260nm kuti tiwerengere kuchuluka kwa nucleic acid, ndiye 1OD imayimira chiyani?
Nucleic acid imakhala ndi nsonga yoyamwa kwambiri pamtunda wa 260nm, womwe uli ndi DNA ndi RNA, komanso zidutswa za nucleic acid (iyi ndiye mfundo yofunika kwambiri).
Mtengo wa OD woyezedwa pamtunda wa 260 nm unalembedwa ngati OD260.Ngati chitsanzocho chili choyera, mtengo wa OD260 ukhoza kuwerengera kuchuluka kwa nucleic acid.
1 OD260=50 μg/ml dsDNA (DNA ya zingwe ziwiri)
=37 μg/ml ssDNA (DNA ya chingwe chimodzi)
= 40 μg/ml RNA
=30 μg/ml dNTPs (oligonucleotides)
Kodi pali kulumikizana kulikonse ndi kusiyana pakati pa RT-PCR, Realtime-PCR ndi QPCR?
RT-PCR ndi yachidule ya Reverse Transcription PCR
Real Time PCR=qPCR, mwachidule cha Quantitative Real Time PCR
Ngakhale Real Time PCR (real-time fluorescent quantitative PCR) ndi Reverse Transcription PCR (reverse transcription PCR) onse akuwoneka kuti amafupikitsidwa ngati RT-PCR.Koma msonkhano wapadziko lonse lapansi ndi wakuti: RT-PCR imatanthawuza kutembenuza PCR.

Kodi nt, bp, ndi kb amagwiritsidwa ntchito bwanji pofotokozera kutalika kwa DNA/RNA mu biology?
nt = nucleotide
bp = base pair base pair
kb = kilobase

Inde, munganene kuti anthu ambiri sasamala za mfundo zazing’onozi!Aliyense amachita izi, ndipo palibe amene angakufunseni kuti ndi chiyani.Mukudziwa kuti izi sizofunikira, sichoncho?

Ayi, Ayi, Ayi, ndikofunikira kwambiri kudziwa izi!chifukwa chani?
Chifukwa mukufuna kutumiza nkhani!M'bale!Kaya mukufuna kumaliza maphunziro anu kapena mukufuna kuchita bwino pa kafukufuku wasayansi, muyenera kudalira zolemba kuti mulankhule!

Kutulutsa kwa nucleic acid kuyenera kukhala kuyesa kosavuta komanso kofunikira kwambiri.Ubwino wa nucleic acid m'zigawo zimadalira mwachindunji zotsatira za mayesero otsatila.

Ngakhale ndanena nthawi zambiri, pali abwenzi ambiri omwe alibe nazo ntchito.Panthaŵiyi ndinaganiza zochoka m’nkhaniyo!

Chidziwitso Chochepa Chofalitsa Mayeso a Quantitative Real-Time PCR, otchedwa MIQE, ndi malangizo oyesera a fluorescence quantitative omwe akhazikitsidwa padziko lonse lapansi, omwe akupereka miyezo yochepa ya chidziwitso choyesera chofunikira powunika kuyesa kwa fluorescence quantitative PCR ndi kusindikiza nkhani.Kupyolera m'mikhalidwe yoyesera ndi njira zowunikira zomwe zimaperekedwa ndi woyesera, owunikirawo akhoza kuwunika bwino kutsimikizika kwa chiwembu choyesera chofufuza.

Zitha kuwoneka kuti mu gawo la nucleic acid m'zigawo, zinthu zotsatirazi zaperekedwa:

"E" ikuwonetsa zomwe ziyenera kuperekedwa ndipo "D" ikuwonetsa zomwe ziyenera kuperekedwa ngati kuli kofunikira.

Fomuyi ndi yovuta kwambiri, kwenikweni, ndikufuna kunena kuti aliyense ayenera kuyambira

kuyera (D), zokolola (D), kukhulupirika (E) ndi kusasinthasintha (E) kuti muwunikire ma Nucleic acid muzinthu zinayi izi.

Malinga experimental zizolowezi, choyamba kulankhula za kuunikira njira chiyero ndi ndende.

Kuyeza kwa OD ndiye njira yomwe amakonda komanso yosavuta yodziwira kwa oyesera.Ponena za mfundoyi, sindifotokoza mwatsatanetsatane apa.Ma laboratories ambiri tsopano amagwiritsa ntchito ma ultra-micro spectrophotometers kuti aunike mwachindunji zitsanzo za nucleic acid.Pomwe ikuwonetsa kuchuluka kwa kuyamwa, pulogalamuyi imapereka mwachindunji mtengo wokhazikika (nucleic acid, mapuloteni ndi utoto wa fulorosenti) ndi ma ratios ogwirizana.Ponena za kuwunika kwa mtengo wa OD, Sungani chithunzichi ndipo mukhala bwino.

Mndandanda wa mayankho a Universal OD

Komabe, pali zochenjeza zingapo zomwe ziyenera kutulutsidwa padera kwa inu.

(Kupatula apo, ndikudziwa kuti muyenera kukhala omwe mumasunga ndikudikirira mpaka mutawafuna!)

Note 1 Zida

Mtengo wa OD udzakhudzidwa ndi zida zosiyanasiyana.Malingana ngati OD260 ili mkati mwamtundu wina, mfundo za OD230 ndi OD280 ndizotanthauzo.Mwachitsanzo, kuyamwa kwa Eppendorf D30 wamba pa 260nm ndi 0 ~ 3A, ndipo NanoDrop One ya Thermo ili pa 260nm.Kuchuluka kwa mpweya ndi 0.5-62.5A.

Note 2Dilution reagent

Mtengo wa OD ukhoza kukhudzidwa ndi kuchepetsedwa kwa ma reagents osiyanasiyana.Mwachitsanzo, kuwerenga kwa OD260/280 kwa RNA yoyeretsedwa mu pH7.5 10mM Trisbuffer ili pakati pa 1.9-2.1, pomwe ili mkatindale amadzimadzi njirachiŵerengero adzakhala m'munsi, mwina 1.8-2.0 okha, koma izi sizikutanthauza kuti khalidwe la RNA kusintha Difference.

Note 3Zinthu zotsalira

Kukhalapo kwa zinthu zotsalira kudzakhudza kulondola kwa muyeso wa ndende ya nucleic acid, kotero ndikofunikira kupewa mapuloteni, phenol, polysaccharide ndi polyphenol zotsalira mu nucleic acid zitsanzo momwe mungathere.

Komabe, kwenikweni, m'zigawo ndi organic reagents ndi njira yakale.Mu zida zamalonda, zotsatira za m'zigawo zimatha kutheka kudzera mzati ya silika yochokera ku adsorption kuphatikiza ndi centrifugation, kupewa ma reagents oopsa komanso owopsa a organic omwe ndi ovuta kuchotsa, etc. Vuto, mongaForegene's nucleic acid extraction kit, sagwiritsa ntchito DNase/RNase ndi poizoni organic reagents panthawi yonse yogwira ntchito, mwachangu komanso motetezeka., ndizotsatira ndizabwino(mwangozi ananena kuti ndi dazi, koma ndikudziwa mukufuna kudziwa).

Chitsanzo 1: Genomic DNA m'zigawo zokolola ndi chiyero

Foregene Soil DNA Isolation Kit (DE-05511) imasamalira zitsanzo za nthaka kuchokera kuzinthu zosiyanasiyana, ndipo kuchuluka ndi kuyera kwa DNA ya genomic zopezedwa zikuwonetsedwa patebulo ili:
Chitsanzo 2: Tissue RNA m'zigawo zokolola ndi chiyero

Animal Total RNA Isolation Kit (RE-03012) inakonza zitsanzo zosiyanasiyana za minofu, ndipo kuchuluka ndi chiyero cha RNA chopezedwa zikuwonetsedwa patebulo ili m'munsimu (kwa minofu ya mbewa):
Komabe, musaganize kuti mwatha ndi mtengo wa OD.Kodi muli ndi zosamalira mfundo zazikuluzikulu zomwe ndakulemberani kutsogolo?

Zindikirani

Ma molekyulu a nucleic acid ogawika adzawerengedwanso mu absorbance.Pongoganiza kuti muli ndi zotsalira za DNA mu RNA, mtengo wanu wa OD udzawoneka ngati wapamwamba kwambiri, koma chiwerengero chenicheni cha RNA sichingadziwike.Kaya RNA yanu ndi Sizikudziwika ngati pali kuwonongeka, kotero tikufunikirabe njira yowunikira kuti tipereke chiweruzo cholondola, ndiko kuti, nucleic acid integrity evaluation yotchulidwa mu MIQE.