I-Buccal Swab/FTA Card DNA Isolation Kit Genomic DNA Extraction noma Ikhithi Yokuhlanzwa ku-Buccal Swabs
Incazelo yekhithi:
Hlanza ngokushesha i-DNA yezinga eliphezulu ye-genomic kusuka kumasampula e-buccal swab/FTA Card.
◮Akukho ukungcoliswa kwe-RNase:Ikholomu Ye-DNA Kuphela ehlinzekwa ikhithi yenza kube nokwenzeka ukususa i-RNA ku-DNA ye-genomic ngaphandle kokwengeza i-RNase phakathi nokuhlolwa, ukuvimbela ilabhorethri ukuthi ingangcoliswa i-RNase yangaphandle.
◮Isivinini esisheshayo:I-Foregene Protease inomsebenzi ophezulu kunama-protease afanayo, futhi igaya amasampula ezicubu ngokushesha;ukusebenza kulula, futhi ukusebenza kwe-genomic DNA extraction kungaqedwa phakathi kwemizuzu engu-20-80.
◮Kulula:I-centrifugation yenziwa ekamelweni lokushisa, futhi asikho isidingo se-centrifugation yezinga lokushisa eliphansi elingu-4°C noma imvula ye-ethanol ye-DNA.
◮Ukuphepha:Akukho kukhishwa kwe-organic reagent edingekayo.
◮Ikhwalithi ephezulu:I-DNA ye-genomic ekhishiwe inezicucu ezinkulu, ayikho i-RNA, ayikho i-RNase, nokuqukethwe kwe-ion ephansi kakhulu, okungahlangabezana nezidingo zokuhlola okuhlukahlukene.
◮Isistimu ye-Micro-elution:Ingakhuphula ukugxila kwe-genomic DNA, elungele ukutholwa komfula noma ukuhlolwa.
Incazelo
Le khithi inikeza indlela ephumelelayo nesheshayo yokuthola i-DNA ye-genomic ephezulu yokugxila kuma-buccal swabs kanye ne-FTA Card (amabala egazi).Ukusebenzisa inkampani yethu's eyingqayizivele ye-DNA-kuphela kwe-silica membrane spin ikholomu kanye nefomula, kuhlanganiswe ne-Foregene Protease, i-high-concentration, i-DNA yekhwalithi ephezulu ye-genomic ingakhishwa emizuzwini engu-80.Ikholomu encane yokuhlanza eklanywe ngokukhethekile ihlanganisa i-DNA ye-genomic, futhi i-DNA ingakhishwa ngenani elincane (15μl) uhlelo lokukhuphula ukugxila kwe-DNA ye-genomic etholiwe, elungele ukutholwa noma ukuhlolwa komfula.Ikhithi ingacubungula isampula eyodwa noma amaningi ngesikhathi, futhi inqubo yokuhlanza ayidingi ukukhishwa kwezinto eziphilayo njenge-phenol, i-chloroform, kanye ne-isopropanol edla isikhathi noma imvula ye-ethanol, futhi ukusebenza kulula futhi kusindisa isikhathi.
Imininingwane
50 Preps
Izingxenye zekhithi
| Isilondolozi se-ST1 |
| Shintshanisa amasheya ST2 |
| Umugqa we-Acrylamide |
| Isilondolozi se-PW |
| Isilondolozi se-WB |
| Isilondolozi EB |
| I-Foregene Protease |
| Ikholomu Ye-DNA Kuphela |
| Iziyalezo |
Izici&izinzuzo
-Akukho ukungcola kwe-RNase: Ikholomu Ye-DNA Kuphela ehlinzekwa ikhithi yenza kube nokwenzeka ukususa i-RNA ku-DNA ye-genomic ngaphandle kokwengeza i-RNase phakathi nokuhlolwa, ukugwema ilabhorethri ukuthi ingangcoliswa i-RNase yangaphandle.
-Isivinini esisheshayo: I-Foregene Protease inomsebenzi ophezulu kunama-protease afanayo, igaya amasampula ngokushesha;ukusebenza okulula.
-Kulula: I-centrifugation yenziwa ekamelweni lokushisa, futhi asikho isidingo se-centrifugation yezinga lokushisa eliphansi elingu-4°C noma imvula ye-ethanol ye-DNA.
-Ukuphepha: Akukho kukhishwa kwe-organic reagent edingekayo.
-Ikhwalithi ephezulu: I-DNA ye-genomic ekhishiwe inezicucu ezinkulu, ayikho i-RNA, ayikho i-RNase, nokuqukethwe kwe-ion ephansi kakhulu, engahlangabezana nezidingo zokuhlola okuhlukahlukene.
-Isistimu ye-Micro-elution: Ingakhuphula ukuhlangana kwe-DNA ye-genomic, elungele ukutholwa noma ukuhlolwa komfula.
Isicelo sekhithi
Ilungele ukuhlanzwa kwe-DNA ye-genomic kusuka kumasampula alandelayo: ama-buccal swabs, i-FTA Card (amabala egazi).
Isitoreji kanye Nempilo yeshelufu
-Le kit ingagcinwa izinyanga ezingu-12 ngaphansi kwezimo ezomile ekamelweni lokushisa (15-25 ° C);uma idinga ukugcinwa isikhathi eside, ingagcinwa ku-2-8°C.
Qaphela: Uma igcinwe ezingeni lokushisa eliphansi, isixazululo sithambekele ekubeni nemvula.Ngaphambi kokusetshenziswa, qiniseka ukuthi ubeka ikhambi kukhithi ekamelweni lokushisa isikhathi esithile.Uma kunesidingo, yishise ngaphambili emanzini okugeza angu-37 ° C imizuzu engu-10 ukuze uhlakaze i-precipitate, futhi uyixube ngaphambi kokusetshenziswa.
-Isixazululo se-Forgene Protease sinefomula eyingqayizivele, esebenzayo uma igcinwe ekamelweni lokushisa isikhathi eside (izinyanga ezingu-3);umsebenzi wayo nokuzinza kuzoba ngcono uma igcinwe ku-4 ° C, ngakho-ke kunconywa ukuyigcina ku-4 ° C, khumbula ukuthi ungayigcini ku -20 ° C.
Ikholomu yokuhlanza ivalekile
Kule kit, ekusebenzeni kokukhipha i-DNA ye-genomic, ikholomu yokuhlanza ikhangiswa ngokuqondile kungxube yesampula ye-enzymatic lysis ngaphandle kwesinyathelo se-centrifugation, futhi ikholomu yokuhlanza ingase ivinjwe ngenxa ye-enzymization engaphelele kanye ne-viscosity ephezulu yesampula.
Izimbangela ezilandelayo zingaba kanje:
1. Ukugaya okungaphelele kwe-enzymatic kwamasampula ezicubu.
Isincomo: Isikhathi sokucubungula isampula se-Foregene Protease singanwetshwa ngokufanelekile noma i-supernatant ingathathwa ngemva kwe-centrifugation ngo-12,000 rpm (~13,400 × g) imizuzu emi-5.
2. Ukusetshenziswa ngokweqile kwamasampula ezicubu noma izicubu ezinkulu.
Isincomo: Kungcono ukuthi ungadluli i-1 Buccal swab kusampula;uma isampula inkulu kakhulu, khulisa umthamo we-Buffer ST1, Foregene Protease, buffer ST2 ngokufanele.
3. I-viscosity yesampula iphezulu kakhulu.
Isincomo: Amasampuli angahlanjululwa ngokufanelekile ngo-10 mM we-Tris-HCl ngaphambi kokukhipha i-genomic DNA.
4. Izingcezwana zekhadi legazi zimunyiwe.
Okutuswayo: Isikhathi esidlulayo se-centrifugation sesinyathelo sesi-6 sendawo yegazi (Ikhadi le-FTA) lokukhishwa kwe-genomic singanwetshwa ngokufanelekile.
Isivuno esiphansi noma ayikho i-DNA
Kuvame ukuba nezinto ezihlukahlukene ezithinta isivuno se-genomic DNA, okuhlanganisa imvelaphi yesampula, izimo zokugcinwa kwesampula, ukulungiswa kwesampula, ukukhohlisa, njll.
I-Genomic DNA ayikwazi ukutholwa ngesikhathi sokukhishwa
Izimbangela ezingenzeka zimi kanje:
1. Ukulondolozwa okungafanele kwamasampuli noma ukugcinwa isikhathi eside kuholela ekonakaleni kwe-DNA ye-genomic.
Okutuswayo: Ama-swabs omlomo kufanele athathwe njengesampula entsha, futhi akufanelekile ukusebenzisa ama-swabs alondoloziwe ukuze kwenziwe imisebenzi yokukhipha i-DNA ye-genomic;amasampula amachashaza egazi kufanele aqinisekise ukuthi ikhwalithi ifanelekile futhi isikhathi sokugcina akufanele sibe side kakhulu.
2. Ukusetshenziswa kwezicubu ezincane kakhulu kungase kuphumele ekukhishweni kwe-DNA ye-genomic ehambisanayo.
Okutuswayo: Landela imiyalelo yesampula ye-buccal swab kumhlahlandlela wokusebenza, futhi usule izikhathi eziningi ngangokunokwenzeka ukuze amaseli anele anamathiselwe ku-swab yomlomo ukuze kukhishwe i-genomic DNA;ukuze kukhishwe isampula yendawo yegazi, indawo yokusika indawo yegazi ingakhuliswa ngokufanelekile.
3. I-Foregene Protease ayilondolozwa ngendlela engafanele, okuholela ekwehleni komsebenzi wayo noma ukungasebenzi.
Okutuswayo: Qinisekisa imibandela yokugcina ye-Foregene Protease noma faka esikhundleni sayo i-Foregene Protease entsha yokusabela kwe-enzymatic.
4. Ukulondolozwa okungalungile kwekhithi noma isikhathi sokugcina side kakhulu, okuholela ekuhlulekeni kwezinye izingxenye zekhithi.
Isincomo: Thenga ikhithi entsha yokuhlukanisa i-Buccal swab DNA ngezinqubo ezihlobene.
5. I-Buffer WB ayifaki i-ethanol ephelele.
Isincomo: Qinisekisa ukuthi isigcinalwazi i-WB sengeza ivolumu elungile ye-ethanol ephelele.
6. I-eluent ayifakwanga kahle kwifilimu ye-silicone.
Okutuswayo: Engeza amaconsi angu-65 °C afudunyezwe ngaphambili phakathi nolwelwesi lwe-silicone bese ushiya ekamelweni lokushisa imizuzu emi-5 ukuze ukwandise ukusebenza kahle kwe-elution.
I-DNA ye-genomic enesivuno esincane ihlukanisiwe
Izimbangela ezilandelayo zingaba kanje:
1. Ukulondolozwa okungafanele kwamasampuli noma ukugcinwa isikhathi eside kuholela ekonakaleni kwe-DNA ye-genomic.
Okutuswayo: Ama-swabs omlomo kungcono athathwe amasampula amasha, futhi ama-swabs alondoloziwe akufanele asetshenziselwe ukukhipha i-DNA ye-genomic.
2. Uma inani lesampula lethishu lilincane kakhulu, okuqukethwe kwe-DNA ekhishiwe kuzoba kuncane.
Okutuswayo: Landela imiyalelo yesampula ye-oral swab kumhlahlandlela wokusebenza, wesula izikhathi eziningi ngangokunokwenzeka ukuze amaseli anele anamathiselwe ku-oral swab ukuze kukhishwe i-genomic DNA.
3. I-Foregene Protease ayilondolozwa ngendlela engafanele, okuholela ekwehleni komsebenzi wayo noma ukungasebenzi.
Okutuswayo: Qinisekisa imibandela yokugcina ye-Foregene Protease noma faka esikhundleni sayo i-Foregene Protease entsha yokusabela kwe-enzymatic.
4. Izinkinga ze-Eluent.
Isincomo: Sebenzisa i-Buffer EB ukuze uthole i-lution;uma usebenzisa i-ddH2O noma ezinye izibonelo, qinisekisa ukuthi i-pH ye-eliate iphakathi kuka-7.0-8.5.
5. I-eluate ayifakwanga ngendlela efanele.
Okutuswayo: Engeza amaconsi alula phakathi nolwelwesi lwe-silicone bese ushiya ekamelweni lokushisa imizuzu emi-5 ukuze ukwandise ukusebenza kahle kwe-elution.
6. Uketshezi lwe-elution lunqwabelana luncane kakhulu.
Isincomo: Sebenzisa i-eluent ukuze uthole i-genomic DNA elution njengoba kudingwa emiyalweni, okungenani kungabi ngaphansi kuka-15 μl.
Ukuhlanzeka okuphansi kwe-genomic DNA kuhlukanisiwe
Ukuhlanzeka okuphansi kwe-DNA ye-genomic kungaholela ekuhlulekeni noma emiphumeleni engagculisi yokuhlola okuya phansi, okufana nalokhu: ama-enzyme awakwazi ukusikwa avuleke, i-PCR ayikwazi ukuthola ucezu lofuzo oluthakaselwayo, njll.
Izimbangela ezingenzeka zimi kanje:
1. Ukungcola kwe-Heteroprotein, ukungcola kwe-RNA.
Ukuhlaziya: Ikholomu yokuhlanza ayizange igezwe kusetshenziswa i-Buffer PW;ikholomu yokuhlanza i-Buffer PW ayizange iwashwe kusetshenziswa isivinini esilungile se-centrifugal.
Okutuswayo: Qinisekisa ukuthi akukho mvula kumandla amakhulu ngaphambi kokwengeza i-ethanol;qiniseka ukuthi ugeza ikholomu yokuhlanza ngokusho kwemiyalelo, futhi lesi sinyathelo asikwazi ukushiywa.
2. Ukungcoliswa kwe-ion engcolile.
Ukuhlaziywa: Ikholomu yokuhlanza i-Buffer WB iye yashiywa noma yawashwa kanye kuphela, okuholele ekungcoleni okusele kwe-ionic.
Okutuswayo: Qiniseka ukuthi uyayigeza i-Buffer WB izikhathi ezi-2 njengoba uyalelwe ukuze ususe ama-ion ayizinsalela ngangokunokwenzeka.
3. Ukungcola kwe-enzyme ye-RNA.
Ukuhlaziya: Ama-RNa angaphandle angeziwe ku-buffer;Ukusebenza kokugeza kwe-Buffer PW bekungalungile, okubangele izinsalela ze-RNase, kukhinyabeze imisebenzi yokuhlola ye-RNA esezansi nomfula, njengokulotshwa kwe-in vitro.
Isincomo: Ikhithi yokuhlukanisa i-nucleic acid yochungechunge lwe-Foregene ingasusa i-RNA ngaphandle kokwengezwa okwengeziwe kwe-RNase, ngaleyo ndlela i-buccal Swab/FTA Card DNA Isolation Kit akudingeki ingeze i-RNase;qiniseka ukuthi ulandela imiyalelo yekholomu yokuhlanza i-Buffer PW, futhi lesi sinyathelo asinakushiywa.
4. Izinsalela ze-Ethanol.
Ukuhlaziywa: I-Buffer WB ayizange yenze i-tube centrifugation engenalutho ngemva kokugeza ikholomu yokuhlanza.
Isincomo: Yenza umsebenzi olungile we-tube centrifugation ngokulandela imiyalelo.
5. Okunye ukungcola kokungcola.
Ukuhlaziya: Amasampuli alondoloziwe noma amasampuli akhethekile awaphathwa kusengaphambili.
Okutuswayo: Yilungise kahle isampula njengoba uyaliwe.
