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Ikhithi yokuhlola ye-Dnase (Fluorescence) HCP0034A Isithombe Esifakiwe
  • Ikhithi yokuhlola ye-Dnase (Fluorescence) HCP0034A

Ikhithi yokuhlola ye-Dnase (Fluorescence)


Inombolo yekati:HCP0034A

Iphakheji: 48T/96T

Ikhithi yokuthola i-DNase isuselwe ocwaningweni lwe-DNA olunelebuli ye-fluorophore.

Incazelo Yomkhiqizo

Idatha yomkhiqizo

Ikhithi yokuthola i-DNase isuselwe ocwaningweni lwe-DNA olunelebuli ye-fluorophore.Uma isampula ingenawo umsebenzi we-DNase, i-probe izinzile futhi ayikhiqizi isignali ye-fluorescent;uma isampula iqukethe umsebenzi we-DNase, i-probe yonakaliswa, okuholela esignali ye-fluorescence ethuthukisiwe kancane kancane;izinga lokukhuphuka kwesignali ye-fluorescence lihlotshaniswa kahle nenombolo kanye nomsebenzi wama-enzyme.Sebenzisa isifundi se-fluorescence microplate ukuze ulinganise kubude begagasi be-ex/em=485/525nm ukuze unqume ukuthi isampula lingcoliswe yi-DNase.


  • Okwedlule:
  • Olandelayo:

  • Isicelo

    Le khithi isetshenziselwa ukuthola ukungcoliswa kwe-DNase kumasampuli.

     

    Cabaphikisayo

    Igama

    I-HCP0034A-01

    (192T)

    I-HCP0034A-02

    (48T)

    10×isixazululo sokusabela

    2.0mL

    0.5mL

    I-DNA probe

    1 ithubhu

    1 ithubhu

    Ibhafa ye-TE

    2.0mL

    0.5mL

    I-DNase I ejwayelekile (2U/μL)

    20μL

    10μL

    I-Standard Dilution Buffer

    12mL

    6ml

    Amanzi angenayo i-DNase

    25ml

    25ml

    DNase RNase kude

    50ml

    50ml

     

    Isitoreji nokuzinza

    1.Kuthuthwe ngo -25 ~ - 15 ℃;

    2.Izingxenye ezihlukene zekhithi zigcinwa ngokuhlukana ngokuvumelana nezinga lokushisa:

    Igama

    izinga lokushisa

    10×isixazululo sokusabela

    -25 ~ -15 ℃

    I-DNA probe

    -25 ~ -15 ℃

    Ibhafa ye-TE

    -25 ~ -15 ℃

    I-DNase I ejwayelekile (2U/μL)

    -25 ~ -15 ℃

    I-Standard Dilution Buffer

    -25 ~ -15 ℃

    Amanzi angenayo i-DNase

    -25 ~ 30℃

    DNase RNase kude

    2 ~ 30℃

    1. Gcina ikhithi engavuliwe izinyanga eziyi-12.

    2.Gcina ikhithi izinyanga eziyi-6 ngemuva kokuvula.Kunconywa ukuthi u-aliquot isixazululo se-DNA probe ngokwenani elilodwa lokusetshenziswa ukuze ugweme ukukhanya nokuqhwa okuphindaphindiwe nokuncibilika.

     

    Imishini edingekayo nezinto ezisetshenziswayo

    1.Isifundi se-fluorescence microplate (kuhlanganise ne-ex/em=485/525nm wavelength)

    2.Amapayipi namathiphu we-DNase&RNase-free

    3.Ishubhu ye-EP ye-DNase&RNase-free

    4.Ipuleti le-DNase&RNase-free elimnyama elingabonisi ngale 96-well

    Ukulungiswa kwe-reagent

    1.Khipha ikhithi bese ulinganisa nezinga lokushisa legumbi (18~25℃), unyakazisa futhi uxube izingxenye ezifana nesixazululo se-reaction esingu-10, i-TE buffer, i-DNase I standard (2U/μL), i-Standard Dilution Buffer, bese i-centrifuge ngokushesha.(Centrifuge at 4000 ~ 7000rpm imizuzwana engu-10).

    2.I-Centrifuge i-DNA probe ku-4000 ~ 7000rpm imizuzwana engu-60 ukuyiqoqa phansi kweshubhu, uvule ngokucophelela i-tube cap, bese wengeza i-40μL TE buffer ukuze incibilike njengesixazululo se-DNA probe storage, aliquot isisombululo sokugcina i-DNA probe ngokusho ukusetshenziswa okukodwa bese uwagcina ku-25 ~ -15 °C ukuze ugweme ukuqhwa okuphindaphindiwe nokuncibilika.Khipha isisombululo sokulondoloza uphenyo ngaso sonke isikhathi uma uhlola, sihlambulule izikhathi ezingu-50 ngebhafa ye-TE (isibonelo, engeza i-490μL TE buffer ku-12μL DNA probe) njengophenyo lwe-DNA olusebenzayo Isixazululo.Gcina yonke ingxenye ye-DNA probe isebenza Isixazululo ku-25 ~ -15 °C ukuze ugweme ukukhanya nokuqhwa okuphindaphindiwe nokuncibilika.

     

    Izinyathelo zokutholwa

    1.Isinyathelo sokusetha inzuzo efanele ngaphambi kokuhlolwa kokuqala, ukugwema ingcuphe yokulahlekelwa ukuzwela noma ukugcwala ngokweqile kwesignali.

    1) amapharamitha wensimbi:

    Ukugubha ipuleti 10 ~ 15s ngaphambi kokuthola;

    Ubude begagasi obujabulisayo λEx=485nm;

    Ubude begagasi lokukhishwa λEm=525nm;

    Sebenzisa umsebenzi wokuzuza othomathikhi;

    Izinga lokushisa 37℃;

    Imodi yephoyinti lokugcina.

    Setha inzuzo kusikali esizenzakalelayo uma kungenzeka, esikhundleni salokho sebenzisa isilungiselelo senzuzo emaphakathi ekuqaleni.

    Qaphela: indlela yokusetha yamathuluzi ahlukene ayihambisani, sicela uthintane nomnikezeli wezinsimbi ukuze uthole imininingwane.

    2) Khetha imithombo emi-2 epuleti lomthombo ongu-96, engeza isixazululo sokusebenza se-DNA engu-10μL kanye ne-10μL 10×isixazululo sokusabela emthonjeni ngamunye;

    3) Engeza u-80μL wamanzi angenayo i-DNase&RNase emthonjeni owodwa, bese wengeza u-79μL wamanzi angenayo i-DNase&RNase kanye no-1μL DNase I standard (2U/μL) komunye umthombo.

    4) Beka ipuleti endaweni emnyama ku-37 ° C bese uyivivinya ngemva kwemizuzu engu-30.

    5) Uma isetha inzuzo kusikali esizenzakalelayo, inani le-Gain lizoboniswa kubha yepharamitha yethuluzi lefayela ledatha, elichazwa ngokuthi G1.

    6) Uma usebenzisa isilungiselelo senzuzo emaphakathi ekuqaleni, kufanele kuqashelwe ukuthi: uma inani eliphakeme le-fluorescence lidlula umkhawulo ongaphezulu wethuluzi, inani lenzuzo kufanele lehliswe ngokufanelekile;uma inani eliphakeme le-fluorescence lingaphansi kakhulu komkhawulo ongaphezulu wethuluzi, inani lenzuzo kufanele lenyuswe ngokufanelekile;Ekugcineni, kutholwa inani elifanele lenzuzo, elichazwa njenge-G2.

     

    2.Setha amapharamitha wensimbi:

    Ukugubha ipuleti 10 ~ 15s ngaphambi kokuthola;

    Ubude begagasi obujabulisayo λEx=485nm;

    Ubude begagasi lokukhishwa λEm=525nm;

    Setha inani lenzuzo ku-G1 noma i-G2 engene kusinyathelo1;

    Izinga lokushisa 37℃;

    Uma umfundi we-microplate esekela imodi ye-kinetic, kunconywa ukusebenzisa imodi yokuthola i-kinetic, ngesikhawu se-1 kuya kumaminithi angu-1.5, futhi isikhathi esiphelele imizuzu engu-30.

     

    3.Ukulungiselela isampula

    Ivolumu yesampula enconyiwe ngu-80μL.Uma isampula elizohlolwa lingaphansi kuka-80μL, nciphisa libe ngu-80μL ngamanzi angenayo i-DNase&RNase.

    Uma isampula elizohlolwa liqukethe izinto ezithinta ukukhanya kwe-fluorophore (njengezixazululo ezimnyama, izinto ze-viscous ezigxiliswe kakhulu noma ama-surfactants), isampula kufanele lihlanjululwe ngamanzi angenayo i-DNase&RNase, kodwa sicela uqaphele ukuthi ukusebenza kokuhlanjululwa kuzothinta ukuzwela.Ukuze isampuli ihlolwe equkethe i-DNase activity inhibitors (njengezixazululo zamandla e-ionic aphezulu, i-pH<4 noma i-pH>9 buffers, ama-protein denaturants, njll.), umphumela wokulinganisa umsebenzi we-enzyme usuwonke wesixazululo sesampula, hhayi umsebenzi ngamunye we-enzyme.

    Nciphisa okujwayelekile kwe-DNase I (2U/μL) Ngesibhafa Esijwayelekile Sokuhlambulula Ngokulandelayo:

    Cha.

    Inqubo yokulungiselela

    Ukugxila

    1

    2μL DNase I standard + 198μL Standard Dilution Buffer

    2×10-2U/μL

    2

    2μL No. 1 isampula + 198μL Standard Dilution Buffer

    2×10-4U/μL

     Nciphisa isampuli engu-No. 2 nge-DNase namanzi angenayo i-RNase izikhathi ezingu-10:

    3

    20μL No. 2 isampula + 180μL DNase&RNase-free water

    2×10-5U/μL

    Inombolo yesampula ye-3 isetshenziswa njengokulawula okuhle;Amanzi angenayo i-DNase&RNase asetshenziswa njengokulawula okungalungile.

    1. Umthamo nokuhlolwa

    1) Engeza isixazululo esisebenzayo se-10μL DNA probe kanye nesixazululo esingu-10μL 10×Reaction epuleti lomthombo elingu-96.Khetha imithombo emi-4 ukuze wengeze isilawuli esinegethivu nokulawula okuhle ngokulandelana, kanye neminye imithombo ukuze wengeze amasampula azohlolwa.Kunemithombo emi-2 eminingi yesampula ngayinye, u-80μL womthombo ngamunye;

    2) Ngokushesha hlola futhi ufunde inani lesignali ye-fluorescence RFU0 ye-0min.Ngemva kokubekwa ebumnyameni ku-37 ℃ imizuzu engu-30, hlola futhi ufunde inani lesignali ye-fluorescence RFU30 for 30min futhi.Uma imodi ye-dynamic yamukelwa, wonke amasiginali we-fluorescence we-0~30min angafundwa.

     

    Ukuhunyushwa kwemiphumela yokuhlolwa

    Uma i-RFU30≥2×RFU0, kubhekwa ukuthi isampula elizohlolwa lingcoliswe yi-DNase.

    Qaphela: uma isampula elizohlolwa lingcole kakhulu noma liqukethe izinto eziphazamisayo, kungase kwenzeke ukuthi i-RFU0 (isampula okufanele ihlolwe) > RFU0 (ukulawulwa kwekhwalithi enhle) kanye ne-RFU30 (isampula okufanele ihlolwe) < 2 × RFU0 (isampula kufanele ihlolwe) ihlolwe), okuholela ekwahlulelweni okungeyikho okungalungile.Ngalesi sikhathi, isampula elizohlolwa lizohlanjululwa ngaphambilini ngamanzi angenayo i-DNase&RNase, bese liyahlolwa.

     

    Ukutholwa komthamo

    Lapho isampula elizohlolwa lingcolile futhi kudingekile ukwahlulela inani lokuhlushwa le-DNase kusampula, linganqunywa ngezinqubo ezilandelayo:

    Nciphisa okujwayelekile kwe-DNase I (2U/μL) Ngesibhafa Esijwayelekile Sokuhlambulula Ngokulandelayo:

    Cha.

    Inqubo yokulungiselela

    ukugxilisa ingqondo

    1

    2μL DNase I standard +198μL Standard Dilution Buffer

    2×10-2U/μL

    2

    2μL No. 1 isampula +198μL Standard Dilution Buffer

    2×10-4U/μL

    3

    100μL No. 2 isampula+100μL Standard Dilution Buffer

    1×10-4U/μL

    4

    100μL No. 3 isampula+100μL Standard Dilution Buffer

    5×10-5U/μL

    5

    100μL No. 4 isampula+100μL Standard Dilution Buffer

    2.5×10-5U/μL

    6

    100μL No.5 isampula+100μL Standard Dilution Buffer

    1.25×10-5U/μL

    Bese uhlambulula amasampula No. 3 ~ No. 5 nge-DNase namanzi angenayo i-RNase izikhathi ezingu-10:

    7

    20μLNo.3 amasampula+180μL DNase kanye namanzi angenayo i-RNase

    1×10-5U/μL

    8

    20μLNo.4 amasampula+180μL DNase kanye namanzi angenayo i-RNase

    5×10-6U/μL

    9

    20μLNo.5 amasampula+180μL DNase kanye namanzi angenayo i-RNase

    2.5×10-6U/μL

    10

    20μLNo.6 amasampula+180μL DNase kanye namanzi angenayo i-RNase

    1.25×10-6U/μL

    No. 7 ~ No. 10 amasampula asetshenziswa njengamazinga;I-DNase & Amanzi angenayo i-RNase njengesampula yokugxila engu-0.

    Hlola isampula ye-concentration engu-0, amazinga kanye nesampuli engcolisiwe ndawonye ngokuya ngezinyathelo zokutholwa ukuze uthole i-RFU0 ne-RFU30.Bala ∆RFU = RFU30-RFU0, thatha ∆RFU (0 concentration) kanye ∆RFU (okujwayelekile) njengokuhlanganisa kanye nokugxilisa ingqondo kwe-DNase I okujwayelekile njenge-abscissa (ukugxilisa okungu-0 ngu-0), yenza ukufaka umugqa, futhi ubale isibalo esifanelekayo y = izembe + B, futhi i-coefficient yokuxhumanisa r kufanele ibe ≥ 0.99.Letha ∆RFU (isampula engcolisiwe) esilinganisweni njengo-y, bala u-x, futhi uyiphindaphinde ngesampula yokuhlanjululwa kwangaphambili okuphindaphindiwe ukuze uthole inani elilinganiselwe lokuhlushwa lesampula elingcolile.

    Qaphela: Ngenxa yokuguquguquka kwesiginali yensimbi, kungenzeka ukuthi ∆RFU<0, ngalesi sikhathi, ibalwa njenge-∆RFU=0.

     

    Ukusebenza kokutholwa

    1. Umkhawulo wokutholwa:DNase I: 1.25×10-6U/μL

    2.Ukunemba: i-intra batch coefficient yokuhluka ≤ 10%, i-inter batch coefficient yokuhluka ≤ 15%

     

    Ukunaka

    1. Umsebenzi wokwengeza isampula kufanele usheshe ngangokunokwenzeka.Isikhathi eside kakhulu sizothinta ukunemba kokuhlolwa.

    2. Amapharamitha wamathuluzi okulebula ama-enzyme e-fluorescent ahlukile.Setha inzuzo efanele ngaphambi kokuhlolwa kokuqala.

    3. Wonke ama-reagents azonyakaziswa ngokuphelele ngaphambi kokusetshenziswa.Lapho wengeza amasampula, amasampula angeziwe azokwengezwa phansi kwepuleti lelebula le-enzyme kahle ukuze kugwenywe ukuwangeza engxenyeni engenhla yodonga lomthombo.Uma wengeza amasampula, qaphela ukuthi ungachaphazi noma ukhiqize amabhamuza.

    4. Izinga elikukhithi i-DNase I, futhi iyunithi yalo esebenzayo ichazwa ngokuthi leyo yunithi eyodwa ichazwa njengenani le-enzyme elizokwehlisa ngokuphelele i-1 µg ye-pBR322 DNA emizuzwini eyi-10 ku-37°C ku-DNase I Reaction Buffer[ 1].Iyunithi eyodwa ye-DNase I ilingana neyunithi ye-0.3 Kunitz[2].

    5. Ukuze ugweme ukungcoliswa kwe-DNase yangaphandle, i-DNase RNase away ingafuthwa phezu kwetafula lokuhlola, amagilavu ​​nezinye izindawo.Ngemuva kwemizuzu emi-5, zihlanze ngamathawula ephepha ahlanzekile bese wenza imisebenzi yokuhlola elandelayo.

     

     

    Bhala umyalezo wakho lapha futhi usithumelele wona