Igazi Direct PCR Kit

Ukukhulisa ngokushesha isakhi sofuzo ngqo kusetshenziswa igazi njengethempulethi ngaphandle kokukhishwa.

Leli thuluzi lisebenzisa i-anti-inhibitor DNA polymerase yezakhi zofuzo ukuze ikhulise kahle izakhi zofuzo zekhophi eyodwa ku-genome yomuntu. Uhlelo lwe-buffer olulungiselelwe kahle kule kit lusiza i-polymerase ukuthi imelane ngokuqinile nokuvinjelwa kwama-PCR inhibitors, ngaleyo ndlela ingakhulisa ngqo i-DNA isebenzisa amaseli egazi namakhompiyutha njengamathempulethi. Lo mkhiqizo kulula ukusebenza futhi awudingi izinyathelo eziyinkimbinkimbi njengokuhlanzwa kwe-DNA noma isampula yokwelashwa kwangaphambili.
Le khithi inikezwa njenge-2 × MasterMix, futhi ukusabela kungenziwa ngokumane ungeze ithempulethi legazi nezinto ezihambisanayo zokuthola. Ingasetshenziswa kumaseli akhulisiwe ezincelisayo njengabantu, amagundane, izingulube, izinkomo nezinye izinhlobo, kanye ne-fresh noma i-4 ℃ cryopreserved igazi eliphelele, i-anticoagulant (i-EDTA, i-citrate, i-heparin), amahlule egazi akhanyayo namachashazi egazi awomile kugcinwe kumakhadi ezentengiselwano kaWhatman 903 naku-FTA Elute.

Ikati. Cha	Ukupakisha Usayizi
4992529	20 ×l × 100 rxn
4992530	20 ×l × 500 rxn

Thumela i-imeyili kithi

Imininingwane yomkhiqizo

Isibonelo sokuhlola

Imibuzo evame ukubuzwa

Amathegi womkhiqizo

Izici

■ Kulula futhi kuyashesha: Ukukhulisa i-PCR kungenziwa ngqo kusetshenziswa igazi njengethempulethi, ngaphandle kwesidingo sezinyathelo eziyindida zokulungiselela isampula nokukhishwa kwe-DNA.
■ Ukuhlanzeka okuphezulu: Ukweqiwa kwesampula yangaphambi kokwelashwa kanye nezinyathelo zokukhishwa kwe-DNA kungasiza ekugwemeni ukungcoliswa okuphambene kwamasampuli.
■ Ukwenza okuphezulu: Ukuhlonza i-PCR kwamasampula amakhulu kungenziwa ngokuhlanganisa ikhithi namacwecwe e-PCR angama-96/384.
■ Ukuqina kwendawo yonke: Le khithi ingakhulisa kahle izingcezu eziphezulu ze-GC noma izingcezu ezinesakhiwo esiyinkimbinkimbi, kanti ubude be-amplification bungafika ku-5 kb.
Resistance Ukumelana nokucindezela okunamandla: Le khithi ingasetshenziswa ezinhlotsheni ezahlukahlukene namasampula egazi agcinwe ngezindlela ezahlukahlukene.

Izicelo

Imikhiqizo ye-PCR yale khithi iqukethe "A" ekugcineni kwe-3', engasetshenziswa ngqo ekwakheni i-vector cloning. Leli kit lingasetshenziselwa ukukhulisa izingcezwana ze-genomic DNA, ukuhlaziywa kwezakhi zofuzo okuphezulu nokuhlaziywa kwe-genotyping (njengokutholwa kofuzo).

Yonke imikhiqizo ingenziwa ngezifiso i-ODM / OEM. Ngemininingwane,sicela uchofoze i-Customized Service (ODM / OEM)

Langaphambilini I-TIANcombi DNA Lyse & Det PCR Kit

Olandelayo: I-Mouse Tissue Direct PCR Kit

	Kusetshenziswa i-anticoagulation yabantu ye-EDTA njengethempulethi, izakhi zofuzo ezi-4 ezinokuqukethwe okuhlukile kwe-GC zandiswa yi-Blood Direct PCR Kit. Uhlelo lokuphendula lwe-PCR lwaluyi-20 μl, futhi igazi le-1 μl lalisetshenziswa njengethempulethi. M: Umaka II weTIANGEN; 1: Usayizi wezinsalela 1090 bp, okuqukethwe kwe-GC 68.1%; 2: Usayizi wengcezu 1915 bp, okuqukethwe kwe-GC 70.4%; 3: Usayizi wezinsalela 448 bp, okuqukethwe kwe-GC 74.8%; 4: Usayizi weqhekeza 1527 bp, okuqukethwe kwe-GC 61.5%. Imiphumela yokuhlola: I-Blood Direct PCR Kit ingakhulisa ngempumelelo izingcezwana ze-DNA nokuqukethwe kwe-GC ebangeni lama-61.5% -74.8%, okuphakamisa ukuthi iyakwazi ukukhulisa izingcezu ze-GC ephezulu.
	Kusetshenziswa i-anticoagulation yabantu njenge-template, izakhi zofuzo ezi-5 ezinobude obuhlukile (i-ActB, i-Prp, i-DN1.0, i-Hn2.0 ne-Hn4.0) zandiswa yi-Blood Direct PCR Kit. Uhlelo lokuphendula lwe-PCR lwaluyi-20 μl, futhi igazi le-1 μl lalisetshenziswa njengethempulethi. M: Umaka II weTIANGEN; 1-3: 3 amasampula egazi ahlukene; I-NTC: lawula ngaphandle kwama-primers. Imiphumela yokuhlola: I-Blood Direct PCR Kit ingakhulisa izingcezu ngobude obufinyelela ku-4 kb, ziphakamisa ukuthi ziyakwazi ukukhulisa izingcezu ezinde.
	Kusetshenziswa i-anticoagulation yomuntu njenge-template, i-Blood Direct PCR Kit isetshenziselwe ukuthola i-PCR ukuthola amasampula wegazi ahlukile. Uhlelo lokuphendula lwe-PCR lwaluyi-20 μl, futhi igazi le-1 μl lalisetshenziswa njengethempulethi. M: Umaka II weTIANGEN; 1-9: inani lokulayisha igazi lingu-0.1 μl, 0.2 μl, 0.3 μl, 0.4 μl, 1 μl, 2 μl, 3 μl, 4 μl no-5 μl, ngokulandelana; I-NTC: ukulawula ngaphandle kwesifanekiso Imiphumela yokuhlola: I-Blood Direct PCR Kit inokumelana okuqinile negazi futhi ingakhulisa amasampuli egazi ngobubanzi bokulayisha obungu-0.1-5 μl.
	Amasampuli egazi avela kumuntu, igundane, inkukhu nezinye izinhlobo ezinemithi ehlukene asetshenziswa njengezifanekiso. I-Blood Direct PCR Kit isetshenziselwe ukukhulisa i-PRNP (yabantu, i-750 bp), i-Actin (i-rat, i-200 bp), ne-β-Actin (Inkukhu, i-1.0 kb). Uhlelo lokuphendula lwe-PCR lwaluyi-20 μl, futhi igazi le-1 μl lalisetshenziswa njengethempulethi. M: Umaka II we-TIANGEN. Imiphumela yokuhlola: Igazi le-PC Direct Kit lingasetshenziswa kumasampula anhlobonhlobo, futhi ukutholwa okuqondile kwe-PCR kungenziwa kumasampula egazi ezinhlotsheni ezahlukahlukene ezinokwelashwa okuhlukile.

Q: Awekho amabhendi wokukhulisa

Isifanekiso se-A-1

■ Ithempulethi iqukethe ukungcola kwamaprotheni noma ama-Taq inhibitors, njll. - —Purify DNA template, susa ukungcola kwamaprotheni noma ukhiphe i-DNA template ngamakhithi okuzihlanza.

■ Ukwehlukaniswa kwethempulethi akuphelele ——Kwandisa ngokufanele ukushisa kwama-denaturation futhi kwandise isikhathi se-denaturation.

■ Ukuwohloka kwesifanekiso ——Lungisa kabusha ithempulethi.

I-A-2 Primer

■ Izinga eliphansi lama-primers ——Re-synthesize the primer.

■ Ukuwohloka kwe-Primer ——Faka ama-primer primer aphezulu abe yivolumu encane yokulondolozwa. Gwema ukubanda okuningi nokuncibilikisa noma ukugcina isikhathi eside u-4 ° C.

■ Idizayini engalungile yama-primers (isb. Ubude be-primer abwenele, i-dimer eyakhiwe phakathi kwama-primers, njll.) -Redesign primers (gwema ukwakheka kwe-primer dimer nesakhiwo sesibili)

I-A-3 Mg²⁺ukuhlushwa

■ Mg²⁺ ukuminyana kuphansi kakhulu —— Khulisa ngokufanele uMg²⁺ukuhlushwa: Lungiselela iMg²⁺ ukugxila ngochungechunge lokuphendula kusuka ku-1 mM kuye ku-3 mM ngesikhawu esingu-0.5 mM ukuthola iMg efanelekile²⁺ ukugxila kuthempulethi ngayinye nakuqala.

Ukushisa kwe-A-4 Annealing

The Ukushisa okuphezulu kokunciphiswa komthelela kuthinta ukubopha kwe-primer ne-template. - - Nciphisa ukushisa okunciphisayo futhi wenze ngcono isimo nge-gradient ka-2 ° C.

Isikhathi se-A-5 Sesandiso

■ Isikhathi esifushane sokunweba — - Khulisa isikhathi sokunweba.

Umbuzo: Ukuthi kunamanga

I-Phenomena: Amasampula amabi nawo akhombisa amabhendi wokulandelana okuqondiwe.

Ukungcola kwe-A-1 kwe-PCR

■ Ukungcola kwesiphambeko sokulandelana okuqondiwe noma imikhiqizo yokukhulisa amandla ——Nakekela ukuthi ungafaki isampuli ngesampula equkethe ukulandelana kwethagethi kusampula engeyinhle noma uchithe iphume kushubhu le-centrifuge. Ama-reagents noma okokusebenza kufanele kwenziwe ngokuzenzakalela ukuqeda ama-nucleic acid akhona, futhi ubukhona bokungcola kufanele kunqunywe ngokuhlolwa kokulawulwa okungalungile.

■ Ukungcola okuyisenzo ——Faka ama-reagents bese uwagcina lapho kushisa okuncane.

I-A-2 Primer

■ Mg²⁺ ukuminyana kuphansi kakhulu —— Khulisa ngokufanele uMg²⁺ ukuhlushwa: Lungiselela iMg²⁺ ukugxila ngochungechunge lokuphendula kusuka ku-1 mM kuye ku-3 mM ngesikhawu esingu-0.5 mM ukuthola iMg efanelekile²⁺ ukugxila kuthempulethi ngayinye nakuqala.

■ Idizayini yokuqala engafanele, nokulandelana kwempokophelo kune-homology ngokulandelana okungeyona eyenhloso. ——Kwakha kabusha ama-primers.

Q: Ukukhulisa okungacacisiwe

I-Phenomena: Amabhendi wokukhulisa we-PCR awahambelani nosayizi olindelekile, kungaba mikhulu noma mincane, noma kwesinye isikhathi womabili amabhendi okukhulisa kanye namaqembu wokukhulisa angaqondile.

I-A-1 Primer

■ Ukucaciswa okungafanelekile kokuqala

——Kwakha kabusha isiqalo.

■ Ukuhlungwa kwe-primer kuphakeme kakhulu — -Kwandisa ngokufanele ukushisa kwe-denaturation futhi kwandise isikhathi se-denaturation.

I-A-2 Mg²⁺ ukuhlushwa

■ UMg²⁺ ukuminyana kuphakeme kakhulu —— Nciphisa kahle ukuhlushwa kwe-Mg2 +: Lungiselela iMg²⁺ ukugxila ngochungechunge lokuphendula kusuka ku-1 mM kuye ku-3 mM ngesikhawu esingu-0.5 mM ukuthola iMg efanelekile²⁺ ukugxila kuthempulethi ngayinye nakuqala.

I-A-3 Thermostable polymerase

■ Inani le-enzyme eyeqile - - Nciphisa inani le-enzyme ngokufanele ngezikhathi ezithile zika-0.5 U.

Ukushisa kwe-A-4 Annealing

■ Izinga lokushisa elinciphisayo liphansi kakhulu ——Kwandisa ngokufanele ithempelesha yokuncisha noma ukusebenzisa indlela yokuncisha izigaba ezimbili

Imijikelezo ye-A-5 PCR

■ Imijikelezo eminingi kakhulu ye-PCR —— Nciphisa inani lemijikelezo ye-PCR.

Q: Ama-Patchy noma ama-smear bands

I-A-1 Primer—— Okucacile okungekuhle—— Yakha kabusha i-primer, shintsha ukuma nobude be-primer ukuthuthukisa ukucaciswa kwayo; noma wenze i-PCR ehlanganisiwe.

I-A-2 Template DNA

——Isifanekiso asihlanzekile —Hlanza ithempulethi noma ukhiphe i-DNA enezikhwama zokuhlanzwa.

I-A-3 Mg²⁺ ukuhlushwa

- —Mg²⁺ ukugxilisa ingqondo kuphakeme kakhulu ——Ukunciphisa kahle uMg²⁺ ukuhlushwa: Lungiselela iMg²⁺ ukugxila ngochungechunge lokuphendula kusuka ku-1 mM kuye ku-3 mM ngesikhawu esingu-0.5 mM ukuthola iMg efanelekile²⁺ ukugxila kuthempulethi ngayinye nakuqala.

I-A-4 dNTP

—— Ukuhlungwa kwama-dNTP kuphakeme kakhulu —— Nciphisa ukuminyana kwe-dNTP ngokufanele

Ukushisa kwe-A-5 Annealing

——Ukushisa okuncishisayo okuphansi impela —— Kukhuphula kahle ukushisa okuncishisayo

Imijikelezo engu-A-6

—Imijikelezo eminingi kakhulu —— Yandisa inani lomjikelezo

Q: Mangaki ithempulethi ye-DNA okufanele ingezwe ku-50 μl system reaction reaction?

Q: Ungazikhulisa kanjani izingcezu ezinde?

Isinyathelo sokuqala ukukhetha i-polymerase efanelekile. I-Taq polymerase ejwayelekile ayikwazi ukufundwa ngenxa yokuntuleka komsebenzi we-3'-5 'wokuxolelwa, futhi ukungafani kahle kuzonciphisa kakhulu ukusebenza kwezingcezu. Ngakho-ke, i-Taq polymerase ejwayelekile ayikwazi ukukhulisa ngempumelelo izingcezu ezihlosiwe ezinkulu kune-5 kb. I-Taq polymerase enokuguqulwa okukhethekile noma okunye ukuthembeka okuphezulu kwe-polymerase kufanele kukhethwe ukuthuthukisa ukusebenza kahle kokunwetshwa nokuhlangabezana nezidingo zokukhulisa isiqeshana eside. Ngaphezu kwalokho, ukukhuliswa kwezingcezu ezinde kudinga nokulungiswa okuhambisanayo komklamo wokuqala, isikhathi sokudlulisa isikhathi, isikhathi eseluliwe, i-pH yesikhondlakhondla, njll. Ngokuvamile, ama-primers ane-18-24 bp angaholela esivunweni esingcono. Ukuvikela ukulimala kwethempulethi, isikhathi sokudonswa kwenani elingu-94 ° C kufanele sehliswe lifike kumasekhondi angama-30 noma ngaphansi ngomjikelezo ngamunye, nesikhathi sokukhuphuka kwamazinga okushisa siye ku-94 ° C ngaphambi kokukhulisa amandla kufanele kube ngaphansi kweminithi elilodwa. Ngaphezu kwalokho, ukusetha izinga lokushisa lokunwebeka cishe ku-68 ° C nokuklama isikhathi sesandiso ngokuya ngesilinganiso se-1 kb / min kungaqinisekisa ukukhuliswa okusebenzayo kwezingcezu ezinde.

Q: Ungakuthuthukisa kanjani ukwethembeka kokukhulisa kwe-PCR?

Izinga lephutha lokukhulisa i-PCR lingehliswa ngokusebenzisa ama-polymerase e-DNA ahlukahlukene ngokuthembeka okuphezulu. Kuwo wonke ama-polymerase eTaq DNA atholakele kuze kube manje, i-Pfu enzyme inezinga lephutha eliphansi kakhulu nokuthembeka okuphezulu (bheka ithebula elihlanganisiwe). Ngaphezu kokukhethwa kwe-enzyme, abacwaningi bangaqhubeka banciphise izinga lokuguqulwa kwe-PCR ngokwenza ngcono izimo zokuphendula, kufaka phakathi ukwakheka kwe-buffer, ukuminyana kwe-polymerase esetshenziswayo nokwandisa inombolo yomjikelezo we-PCR.

Bhala umyalezo wakho lapha bese uwuthumela kithi