Quantification ntawm Nucleic Acids hauv Spectrophotometers
Kev ntsuas ntawm nucleic acids yog kev siv ntau dua ntawm tus spectrophotometer. Oligonucleotides, ib leeg-tso tseg, muab ob npaug rau DNA, thiab RNA tuaj yeem ua tau ntau yam hauv buffers. Qhov nqes dej ntawm lub siab tshaj plaws ntawm lub ncov ntawm nucleic acid yog 260 nm. Txhua nucleic acid muaj ib qhov sib txawv ntawm cov khoom sib xyaw thiab thiaj li muaj kev hloov ntau yam. Yuav kom ntsuas ntau hom nucleic acids, xaiv cov coefficients corresponding ua ntej. Piv txwv, qhov absorbance ntawm 1OD yog sib npaug li 50 μg / ml dsDNA, 37 μg / ml ssDNA, 40 μg / ml RNA, thiab 30 μg / ml Olig, feem. Qhov txiaj ntsim ntawm qhov tsis txaus tom qab qhov kev ntsuas tau hloov dua siab tshiab los ntawm cov coefficient saum toj no kom tau txais cov qauv coj los sib piv. Ua ntej kuaj, xaiv qhov txheej txheem kom raug, nkag mus rau tagnrho ntawm cov khoom lag luam thiab kev ua haujlwm, thiab ntsuam xyuas cov ntaub ntawv dawb thiab qauv. Txawm li cas los, qhov kev sim tsis yooj yim. Cov kev nyeem tsis ruaj tsis khov yuav yog qhov mob taub hau loj tshaj rau cov neeg sim. Qhov siab dua qhov cuab yeej ntawm qhov ntsuas, ntau dua qhov uas tau pom tias absorbance drift.
Qhov tseeb, tus qauv tsim thiab kev ua haujlwm ntawm tus qauv spectrophotometer tso cai rau qhov hloov ntawm qhov tsis txaus ntseeg kom hloov tsis dhau ib qho, qhov ntawd yog qhov cuab yeej muaj qhov tseeb thiab qhov tseeb. Xws li Eppendorf Biophotometer qhov tseeb ≤ 1.0% (1A). Cov qhabnias ntawm cov kev xeem ntau sib txawv ntawm qhov nruab nrab ntawm 1.0% thiab yog txhua qhov qub. Tsis tas li ntawd, lub cev nqaij daim tawv ntawm cov nucleic acid, lub pH ntawm cov tsis tuaj yeem rau dissolving cov nucleic acid, ion concentration, thiab lwm yam yuav tsum tau muab xam rau hauv tus account: Thaum lub sij hawm qhov kev sim siab, siab heev ion concentration yuav ua rau nyeem ntawv rau drift li TE, uas yuav zoo heev rau kev nyeem ntawv. Lub diluted concentration ntawm tus qauv kuj yog qhov tsis tseem ceeb: vim yog qhov tsis pom qhov me me ntawm cov qauv me me, tshwj xeeb tshaj yog cov nucleic acid samples. Lub xub ntiag ntawm cov khoom me no cuam tshuam nrog qhov kev kuaj ntawd. Thiaj li txo qee yam ntawm cov kev sib tw, qhov nqus ntawm cov nucleic acid yuav tsum muaj tsawg kawg yog 0.1 A, thiab qhov absorbance zoo li 0.1-1.5 A. Nyob rau hauv qhov no, yuav tsum tsis muaj cov npuas nyob hauv sib tov, thiab tsis muaj kev ncua hauv kev seem. Txwv tsis pub, nyeem cov kev hloov ua nruj ua tsiv; tib xim khob tib yam yuav tsum tau siv los kuaj qhov seem thiab tus qauv; txwv tsis pub, qhov sib txawv ntawm qhov sib txawv yog loj dhau; kev hloov coefficient thiab cov qauv hnyav hnyav yog tib yam; Lub cuvette hnav los ntawm lub qhov rais; qhov ntim ntawm qhov qauv yuav tsum ncav ntau tshaj qhov yuav tsum tau ntim tsawg kawg ntawm xim khob, thiab lwm yam.
Ntxiv nrog rau qhov nucleic acid concentration, tus spectrophotometer qhia tau hais tias ob peb qho tseem ceeb ntawm lub sij hawm ib zaug los qhia qhov purity ntawm tus qauv, xws li A260 / A280 piv, rau lub hom phiaj ntawm kev soj ntsuam cov purity ntawm tus qauv, txij thaum lub siab lub ncov ntawm cov protein yog 280 nm. Cov kab mob ntshiab muaj piv txwv ntau tshaj 1.8 (DNA) lossis 2.0 (RNA). Yog hais tias qhov sib piv yog tsawg dua 1.8 lossis 2.0, nws hais tias muaj protein ntau los yog phenolic tshuaj. A230 qhia tias muaj qee tus kabmob hauv cov qauv, xws li cov carbohydrates, peptides, phenols, etc. Qhov ratio ntawm ntshiab nucleic acid A260 / A230 ntau dua 2.0. A320 pom cov turbidity ntawm kev daws thiab lwm yam cuam tshuam yam. Ntshiab qauv, A320 yog feem ntau 0.
Lub tuam txhab tshwj xeeb hauv kev muag khoom ntawm spectrophotometers , txais tos cov neeg siv khoom uas muaj qhov xav tau los tham nrog thiab yuav khoom, peb yuav muab koj nrog sincere cov kev pab cuam thiab cov khoom zoo, nrog rau cov nqi qis dua.
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