Dnase Assay Kit (Fluorescence)

Daim ntawv thov

Cov khoom siv no yog siv los kuaj pom DNase paug hauv cov qauv.

Ccov omponents

Cia thiab ruaj khov

1.Thauj hauv -25 ~ - 15 ℃;

2.Cov khoom sib txawv ntawm cov khoom siv tau muab cais raws li qhov kub thiab txias:

1. Khaws cov khoom siv tsis tau qhib rau 12 lub hlis.

2.Khaws cov khoom siv rau 6 lub hlis tom qab qhib.Nws raug nquahu kom aliquot DNA sojntsuam tshuaj raws li kev siv ib zaug kom tsis txhob muaj lub teeb thiab rov ua kom khov thiab thawing.

Cov khoom siv thiab cov khoom siv uas yuav tsum tau muaj

1.Fluorescence microplate nyeem ntawv (xws li ex / em = 485 / 525nm wavelength)

2.DNase & RNase-dawb pipettes thiab cov lus qhia

3.DNase & RNase-dawb EP raj

4.DNase & RNase-dawb dub tsis-pob tshab 96-zoo phaj

Kev npaj reagent

1.Tshem tawm cov khoom siv thiab sib npaug rau chav sov (18 ~ 25 ℃), co thiab sib tov cov khoom xws li 10 × tshuaj tiv thaiv, TE tsis, DNase I standard (2U / μL), Standard Dilution Buffer, thiab ces centrifuge tam sim ntawd.(Centrifuge ntawm 4000 ~ 7000rpm rau 10 vib nas this).

2.Centrifuge DNA sojntsuam ntawm 4000 ~ 7000rpm rau 60 vib nas this kom sib sau ua ke rau hauv qab ntawm lub raj, ua tib zoo qhib lub raj cap, thiab ntxiv 40μL TE tsis rau yaj raws li DNA sojntsuam cia tshuaj, aliquot DNA sojntsuam cia tshuaj raws li lub siv ib zaug thiab khaws cia ntawm -25 ~ -15 ° C kom tsis txhob rov khov thiab thawing.Tshem tawm cov kev sojntsuam khaws cia txhua lub sijhawm koj sim, dilute nws 50 zaug nrog TE tsis (piv txwv li, ntxiv 490μL TE tsis nyob rau hauv 12μL DNA sojntsuam) raws li DNA sojntsuam ua haujlwm daws.Khaws cov khoom seem ntawm DNA sojntsuam ua haujlwm daws ntawm-25 ~ -15 ° C kom tsis txhob lub teeb thiab rov ua kom khov thiab thawing.

Kev txheeb xyuas cov kauj ruam

1.Kauj ruam los teeb tsa qhov tsim nyog nce ua ntej qhov kev sim thawj zaug, kom tsis txhob muaj kev pheej hmoo ntawm rhiab heev poob lossis teeb liab oversaturation.

1) Kev ntsuas ntsuas:

Co lub phaj 10 ~ 15s ua ntej kuaj pom;

Excitation wavelength λEx = 485nm;

Emission wavelength λEm = 525nm;

Siv qhov kev nce qib tsis siv neeg;

Kub 37 ℃;

Endpoint hom.

Teem qhov nce rau nws pib-scale yog tias ua tau, hloov pauv siv qhov nruab nrab nce qhov pib.

Nco tseg: txoj kev teeb tsa ntawm cov cuab yeej sib txawv tsis sib xws, thov nrog tus neeg muag khoom ntsuas kom paub meej.

2) Xaiv 2 qhov dej ntawm lub phaj 96-zoo, ntxiv 10μL DNA sojntsuam ua haujlwm thiab 10μL 10 × tshuaj daws rau txhua qhov dej;

3) Ntxiv 80μL ntawm DNase & RNase-dawb dej rau ib lub qhov dej, thiab ntxiv 79μL ntawm DNase & RNase-dawb dej thiab 1μL DNase I standard (2U/μL) rau lwm qhov dej.

4) Muab lub phaj tso rau hauv qhov chaw tsaus ntawm 37 ° C thiab sim tom qab 30 feeb.

5) Yog tias teeb tsa qhov nce mus rau autoscale, Kev nce nqi yuav tshwm sim hauv qhov ntsuas ntsuas qhov ntsuas ntawm cov ntaub ntawv cov ntaub ntawv, suav tias yog G1.

6) Thaum siv qhov nruab nrab nce qhov chaw pib, nws yuav tsum tau muab sau tseg tias: yog tias tus nqi fluorescence siab tshaj qhov txwv ntawm qhov ntsuas, qhov nce nqi yuav tsum raug txo kom tsim nyog;Yog hais tias tus nqi fluorescence siab nyob deb ntawm qhov siab tshaj ntawm qhov ntsuas, qhov kev nce nqi yuav tsum tau nce kom tsim nyog;Thaum kawg, qhov tsim nyog tau txais txiaj ntsig tau txais, suav tias yog G2.

2.Teem lub ntsuas parameter:

Co lub phaj 10 ~ 15s ua ntej kuaj pom;

Excitation wavelength λEx = 485nm;

Emission wavelength λEm = 525nm;

Teem tus nqi nce rau G1 lossis G2 tau txais hauv kauj ruam 1 ;

Kub 37 ℃;

Yog tias tus nyeem ntawv microplate txhawb hom kinetic, nws raug nquahu kom siv hom kev tshawb nrhiav kinetic, nrog lub sijhawm ntawm 1 txog 1.5 feeb, thiab tag nrho lub sijhawm yog 30 feeb.

3.Kev npaj ua qauv

Cov qauv pom zoo yog 80μL.Yog tias cov qauv yuav tsum tau kuaj tsawg dua 80μL, dilute rau 80μL nrog DNase ＆RNase-dawb dej.

Thaum cov qauv mus kuaj muaj cov tshuaj uas cuam tshuam rau lub luminescence ntawm fluorophore (xws li cov kev daws teeb meem tsaus, cov tshuaj muaj kuab paug siab lossis cov tshuaj surfactants), cov qauv yuav tsum tau diluted nrog DNase & RNase-dawb dej, tab sis thov nco ntsoov tias kev ua haujlwm dilution yuav cuam tshuam. qhov rhiab heev.Rau cov qauv kuaj uas muaj DNase kev ua haujlwm inhibitors (xws li cov kev daws teeb meem ionic siab, pH <4 lossis pH> 9 buffers, protein denaturants, thiab lwm yam), qhov ntsuas tau yog tag nrho cov enzyme kev ua ntawm cov qauv tshuaj, tsis yog kev ua haujlwm ntawm tus kheej ntawm enzyme.

Dilute DNase I standard (2U / μL) nrog Standard Dilution Buffer raws li hauv qab no:

 Dilute tus qauv No.2 nrog DNase & RNase-dawb dej rau 10 zaug:

No. 3 qauv yog siv los tswj qhov zoo;DNase & RNase-dawb dej yog siv los tswj tsis zoo.

1. Dosing thiab kuaj

1) Ntxiv 10μL DNA sojntsuam ua haujlwm daws teeb meem thiab 10μL 10 × Reaction tov rau 96-zoo phaj.Xaiv 4 lub qhov dej ntxiv rau qhov kev tswj tsis zoo thiab kev tswj xyuas qhov zoo, thiab lwm lub qhov dej ntxiv rau cov qauv coj mus kuaj.Muaj 2 ntau lub qhov dej rau txhua tus qauv, 80μL rau txhua qhov dej;

2) Tam sim ntawd sim thiab nyeem cov teeb liab fluorescence tus nqi RFU0 rau 0min.Tom qab muab tso rau hauv qhov tsaus ntuj ntawm 37 ℃ rau 30min, sim thiab nyeem cov fluorescence teeb liab nqi RFU30 rau 30min dua.Yog tias siv hom dynamic, tag nrho cov teeb liab fluorescence tuaj yeem nyeem tau 0 ~ 30min.

Kev txhais cov txiaj ntsig kev xeem

Yog tias RFU30≥2 × RFU0, nws raug txiav txim siab tias tus qauv yuav raug kuaj yog kis los ntawm DNase.

Nco tseg: yog tias tus qauv yuav raug kuaj pom muaj kab mob hnyav lossis muaj cov khoom cuam tshuam, nws yuav tshwm sim tau tias RFU0 (cov qauv yuav tsum tau kuaj) > RFU0 (zoo tswj tau zoo) thiab RFU30 (cov qauv yuav tsum tau kuaj) < 2 × RFU0 (piv txwv los ua kuaj), ua rau muaj kev txiav txim tsis zoo.Lub sijhawm no, cov qauv yuav tsum tau kuaj ua ntej yuav tsum tau diluted nrog DNase & RNase-dawb dej, thiab tom qab ntawd sim.

Quantitative nrhiav tau

Thaum cov qauv yuav raug kuaj tsis muaj paug thiab nws yuav tsum txiav txim siab qhov concentration ntawm DNase hauv cov qauv, nws tuaj yeem txiav txim siab los ntawm cov txheej txheem hauv qab no:

Dilute DNase I standard (2U / μL) nrog Standard Dilution Buffer raws li hauv qab no:

Tom qab ntawd dilute No. 3 ~ No. 5 kuaj nrog DNase & RNase-dawb dej rau 10 zaug:

No. 7 ~ No. 10 cov qauv siv raws li cov qauv;DNase & RNase-dawb dej raws li 0-concentration qauv.

Test 0-concentration qauv, cov qauv thiab cov qauv tsis huv ua ke raws li kev kuaj xyuas cov kauj ruam kom tau txais RFU0 thiab RFU30.Calculate ∆RFU = RFU30-RFU0, coj ∆RFU (0 concentration) thiab ∆RFU (tus qauv) raws li kev tswj hwm thiab DNase I concentration ntawm tus qauv raws li abscissa (0 concentration yog 0), nqa tawm linear haum, thiab xam qhov haum kab zauv y = ax + B, thiab correlation coefficient r yuav tsum ≥ 0.99.Nqa ∆RFU (cov qauv tsis huv) mus rau hauv qhov sib npaug li y, xam x, thiab muab nws los ntawm cov qauv ua ntej dilution ntau kom tau txais qhov kwv yees kwv yees tus nqi ntawm cov qauv paug.

Nco tseg: Vim muaj qhov hloov pauv ntawm qhov ntsuas ntsuas, nws yuav tshwm sim tias ∆RFU<0, lub sijhawm no, nws raug suav tias yog ∆RFU = 0.

Kev kuaj xyuas kev ua haujlwm

1. Kev kuaj pom qhov txwv: DNase I: 1.25 × 10-6U / μL

2.Precision: intra batch coefficient ntawm variation ≤ 10%, inter batch coefficient ntawm variation ≤ 15%

Nco ntsoov

1. Cov qauv ntxiv kev ua haujlwm yuav tsum ceev li sai tau.Lub sijhawm ntev dhau lawm yuav cuam tshuam qhov tseeb ntawm qhov kev sim.

2. Cov tsis sib txawv ntawm cov khoom siv fluorescent enzyme labeling txawv.Teem kom tsim nyog nce ua ntej qhov kev xeem thawj zaug.

3. Tag nrho cov reagents yuav tsum tau shaken tag nrho ua ntej siv.Thaum ntxiv cov qauv, cov qauv ntxiv yuav tsum muab ntxiv rau hauv qab ntawm daim ntawv enzyme daim ntawv lo kom tsis txhob muab ntxiv rau sab sauv ntawm phab ntsa dej.Thaum ntxiv cov qauv, xyuam xim tsis txhob txaws lossis tsim npuas.

4. Tus qauv hauv cov khoom siv yog DNase I, thiab nws lub zog ua haujlwm tau txhais raws li ib chav tsev tau txhais raws li tus nqi ntawm cov enzyme uas yuav degrade tag nrho 1 µg ntawm pBR322 DNA hauv 10 feeb ntawm 37 ° C hauv DNase I Reaction Buffer[ 1].Ib chav DNase I yog sib npaug rau 0.3 Kunitz unit[2].

5. Txhawm rau kom tsis txhob muaj cov kab mob exogenous DNase, DNase RNase tam sim no tuaj yeem txau rau ntawm qhov chaw ntawm lub rooj sim, hnab looj tes thiab lwm qhov chaw.Tom qab 5 feeb, ntxuav lawv nrog cov ntaub so ntswg huv thiab tom qab ntawd ua cov kev sim tom ntej.