Spectrophotometers tau dhau los ua cov twj paj nruag niaj hnub siv hauv lub chaw soj nstuam molecular ib. Feem ntau siv rau nucleic acid, protein ntau thiab ntau ntawm cov kab mob kev loj hlob zoo.
Cov ntsiab cai yooj yim ntawm spectrophotometer
Lub tshuab hluav taws xob siv cov khoom siv hluav taws xob uas tuaj yeem tsim tau ntau lub wavelengths, thiab dhau los ntawm cov khoom siv hluav taws xob ntau ntau los tsim cov teeb meem ntawm lub nthwv dej. Tom qab lub teeb hluav taws xob dhau los ntawm cov qauv coj mus kuaj, qee qhov ntawm lub teeb ci tau nqus, thiab cov nqus ntawm cov qauv tau muab xam, yog li hloov mus rau hauv ib qho qauv concentration. Cov. Qhov kev nqus ntawm cov qauv yog sib piv rau cov concentration ntawm cov qauv.
Kev suav ntawm nucleic acids
Qhov kev txiav txim zoo ntawm nucleic acids yog qhov feem ntau siv txoj haujlwm ntawm lub spectrophotometer. Oligonucleotides, ib leeg-tso tseg, muab ob npaug tso tseg DNA, thiab RNA tuaj yeem raug ntsuas tau hauv qhov tsis xws luag. Qhov nqus ntawm qhov siab ntawm qhov siab tshaj plaws ntawm cov ncov dej khov ntawm nucleic acid yog 260 nm. Cov qauv sib xyaw ntawm txhua cov nucleic acid sib txawv, yog li cov ntsiab lus hloov pauv tau sib txawv. Txhawm rau kev txheeb xyuas ntau hom nucleic acids, xaiv cov coefficients sib xyaw ua ntej. Piv txwv li, qhov nqus ntawm 1 OD sib raug rau 50 μg / ml ntawm dsDNA, 37 μg / ml ntawm ssDNA, 40 μg / ml ntawm RNA, thiab 30 μg / ml ntawm Olig. Qhov kev nqus tom qab qhov kev xeem raug hloov pauv los ntawm cov coefficients saud kom tau cov qauv coj ua kom sib haum. Ua ntej kuaj, xaiv cov txheej txheem tsim nyog, nkag mus rau qhov ntim ntawm qhov kev daws teeb meem thiab kev tsis sib haum, thiab tom qab ntawd kuaj cov khoob thiab qauv tshuaj. Txawm li cas los xij, kev sim tsis yeej ib txwm swm. Kev nyeem ntawv tsis ruaj khov yuav ua rau mob taub hau loj tshaj plaws rau tus kws sim. Kev ntsuas nrog qhov siab dua rhiab qhia pom ntau dua kev xau hauv kev nqus.
Qhov tseeb, tus qauv tsim qauv thiab kev ua haujlwm ua haujlwm ntawm lub spectrophotometer tso cai rau kev nqus kom sib txawv los ntawm ntau qhov, uas yog, qhov ntsuas tau muaj qee qhov tseeb thiab qhov tseeb. Piv txwv, qhov tseeb ntawm qhov Eppendorf Biophotometer yog ≤1.0% (1A). Cov txiaj ntsig ntawm kev ntsuas ntau yam sib txawv ntawm kwv yees li 1.0% ntawm qhov nruab nrab thiab qhov qub. Tsis tas li ntawd, nws tseem yuav tau txiav txim siab lub cev lub zog ntawm lub zog nucleic acid nws tus kheej thiab pH ntawm lub ntas dej uas cov nucleic acid yog yaj, cov ion concentration, thiab lwm yam.: Thaum cov ion concentration yog siab dhau thaum kuaj, cov kev hloov pauv nyeem ntawv, yog li nws raug nquahu kom siv qee yam nqi pH thiab lub zog qis. Buffers, xws li TE, zoo heev tswj kev nyeem ntawv. Lub zom zaws ntawm qhov ua piv txwv kuj yog ib qho tseem ceeb uas tsis tuaj yeem tsis quav ntsej: vim muaj kev nruam ntaws ntawm qee qhov zoo, tshwj xeeb tshaj yog nucleic acid cov piv txwv, hauv cov qauv. Lub xub ntiag ntawm cov khoom me me cuam tshuam nrog kev tshuaj ntsuam. Txhawm rau txo cov nyhuv ntawm cov lus hais ntawm cov ntawv twv, qhov kev nqus ntawm nucleic acid yuav tsum muaj tsawg kawg yog 0.1 A, thiab kev nqus tau zoo dua li ntawm 0.1 thiab 1.5 A. Nyob rau hauv cov kab no, cuam tshuam ntawm kev faib tawm yog qhov me me thiab qhov tshwm sim yog ruaj khov.
Qhov no txhais tau hais tias qhov kev ntsuas siab ntawm cov qauv yuav tsum tsis txhob dhau tus siab lossis siab dhau (dhau ntawm qhov ntsuas ntawm qhov ntsuas ntawm lub ntsuas cua). Thaum kawg, cov haujlwm tseem ceeb, xws li kev sib xyaw yuav tsum muaj kom txaus, txwv tsis pub cov nqus dej tsawg dhau, txawm tias cov nqi tsis zoo; cov sib xyaw tsis tuaj yeem muaj npuas, cov kua ntsiag to tsis muaj qhov teeb meem ncua, txwv tsis pub nyeem ntawv tawm zuj zus; tib lub cuvette yuav tsum siv los ntsuas qhov dawb thiab piv txwv Txwv tsis pub, qhov sib txawv hauv kev kub siab yog loj heev; qhov hloov ua lwm yam thiab ntsuas qhov ntsuas cov qauv raug xaiv ua ntu zus; lub cuvette nrog qhov rai hnav tsis tau siv; qhov ntim ntawm tus qauv yuav tsum ncav qhov ntim tsawg kawg uas yuav tsum tau ua los ntawm cuvette.
Ntxiv nrog rau qhov kev tsom xam nucleic acid, lub spectrophotometer tseem qhia pom ntau qhov tseem ceeb qhia tias cov pa huv ntawm qhov piv txwv, xws li qhov sib piv ntawm A 260 / A 280, siv los ntsuas qhov ntshiab ntawm cov qauv vim tias qhov ncov nqus ntawm cov protein yog 280 nm. Tus qauv ntshiab nrog qhov sib piv ntau dua 1.8 (DNA) lossis 2.0 (RNA). Yog tias qhov sib piv tsawg dua li 1.8 lossis 2.0, nws qhia tias muaj cov protein lossis phenolic cov nyob. Ib 230 qhia tau tias qee cov pa paug nyob hauv tus qauv, xws li carbohydrates, peptides, phenols, thiab lwm yam, thiab tus piv ntawm purer nucleic acid A 260 / A 230 yog ntau dua 2.0. A 320 kuaj pom qhov ua tsis haum ntawm kev daws thiab lwm yam cuam tshuam nrog cuam tshuam. Rau cov qauv ntshiab, A 320 feem ntau yog 0.
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