Ikhithi yokuhlola ye-Dnase (Fluorescence)
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.
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.
- 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.