Kit ea PC Direct ea Mali

Ho matlafatsa ka potlako phatsa ea lefutso ka kotloloho ho sebelisa mali e le template ntle le ho ntšoa.

Setsi sena se amohela anti-inhibitor DNA polymerase ea liphatsa tsa lefutso ho matlafatsa ka nepo liphatsa tsa lefutso tsa mofuta o le mong liphatseng tsa lefutso tsa motho. Sisteme e ntlafalitsoeng hantle ea lisebelisoa tsena e thusa polymerase ho hanela ka matla thibelo ea PCR inhibitors, ka hona e ka holisa DNA ka kotloloho e sebelisa lisele tsa mali le tse ntlafalitsoeng joalo ka litempele. Sehlahisoa sena se bonolo ho sebetsa ebile ha se hloke mehato e thata joalo ka tlhoekiso ea DNA kapa sampole ea kalafo ea pele.
Setsi sena se fanoa e le 2 × MasterMix, 'me karabelo e ka etsoa ka ho eketsa feela template ea mali le li-primers tse tsamaellanang. E ka sebelisoa liseleng tse ntlafalitsoeng tsa liphoofolo tse anyesang joalo ka batho, litoeba, likolobe, likhomo le mefuta e meng, hammoho le mali a hloekileng kapa a 4 ℃ cryopreserved, anticoagulant (EDTA, citrate, heparin), mali a mali le mali a omileng e bolokiloe ho likarete tsa khoebo tsa Whatman 903 le FTA Elute.

Katse. Che	Paka Size
4992529	20 ×l × 100 rxn
4992530	20 ×l × 500 rxn

Romela ya imeile ho re

Product Qaqileng

Mohlala oa Teko

LBH

Lihlahisoa tsa Product

Likaroloana

■ E bonolo ebile e potlakile: Kholiso ea PCR e ka etsoa ka kotloloho ho sebelisoa mali joalo ka template, ntle le tlhoko ea methati e tenang ea boitokisetso ba sampole le ho ntšoa ha DNA.
■ Bohloeki bo phahameng: Ho tlola mehlala ea kalafo ea kalafo le mehato ea ho ntša DNA ho ka thusa ho qoba tšilafalo ea mehlala.
■ Phallo e phahameng: Boitsebiso ba PCR ba mehlala e meholo bo ka etsoa ka ho kopanya lisebelisoa le lipoleiti tsa PCR tse 96/384.
■ Bokahohle bo matla: Seteishene sena se ka matlafatsa likaroloana tse phahameng tsa GC kapa likhechana tse nang le sebopeho se rarahaneng sa bobeli, 'me bolelele ba bophahamo e ka ba 5 kb.
■ Ho hanyetsa khatello ea maikutlo ka matla: Setsi sena se ka sebelisoa bakeng sa mefuta e fapaneng le disampole tsa mali tse bolokiloeng ka mekhoa e fapaneng.

Likopo

Lihlahisoa tsa PCR tsa sesebelisoa sena li na le "A" pheletsong ea 3', e ka sebelisoang ka kotloloho bakeng sa ho hlophisoa ha vektara ea TA. Setsi sena se ka sebelisoa bakeng sa ho matlafatsa likhechana tsa DNA ea genomic, tlhahlobo e phahameng ea liphatsa tsa lefutso le tlhahlobo ea genotyping (joalo ka tlhahlobo ea liphatsa tsa lefutso).

Lihlahisoa tsohle li ka fetoloa bakeng sa ODM / OEM. Bakeng sa lintlha,ka kopo tlanya Customized Service (ODM / OEM)

E fetileng: TIANcombi DNA Lyse & Det PCR Kit

E 'ngoe: Lisele tsa Mouse Direct PCR Kit

	Ho sebelisa anticoagulation ea motho ea EDTA e le template, liphatsa tsa lefutso tse 4 tse nang le litaba tse fapaneng tsa GC li ile tsa holisoa ke Blood Direct PCR Kit. Sisteme ea PCR reaction e ne e le 20 μl, mme mali a 1 μl a sebelisitsoe e le template. M: TIANGEN Lesupa II; 1: Sekhechana sa boholo ba 1090 bp, dikahare tsa GC 68.1%; 2: Sekhechana sa boholo ba 1915 bp, GC e na le 70.4%; 3: Sekhechana sa boholo ba 448 bp, dikahare tsa GC ke 74.8%; 4: Sekhechana sa boholo ba 1527 bp, litaba tsa GC ke 61.5%. Liphetho tsa liteko: Blood Direct PCR Kit e ka matlafatsa likhechana tsa DNA ka sehlahisoa sa GC se ka bang 61.5% -74.8%, e fana ka maikutlo a hore e khona ho holisa likhechana tse phahameng tsa GC.
	Ho sebelisa anticoagulation ea motho e le template, liphatsa tsa lefutso tse 5 tse bolelele bo fapaneng (ActB, Prp, DN1.0, Hn2.0 le Hn4.0) li ile tsa holisoa ke Blood Direct PCR Kit. Sisteme ea PCR reaction e ne e le 20 μl, mme mali a 1 μl a sebelisitsoe e le template. M: TIANGEN Lesupa II; 1-3: 3 mehlala e fapaneng ea mali; NTC: taolo ntle le li-primers. Liphetho tsa liteko: Blood Direct PCR Kit e ka holisa likaroloana ka bolelele ho isa ho 4 kb, ho fana ka maikutlo a hore e khona ho holisa likhechana tse telele.
	Ho sebelisa anticoagulation ea motho e le template, Blood Direct PCR Kit e ne e sebelisetsoa PCR ho fumana mefuta e fapaneng ea mali. Sisteme ea PCR reaction e ne e le 20 μl, mme mali a 1 μl a sebelisitsoe e le template. M: TIANGEN Lesupa II; 1-9: palo ea mali e laoloang ke 0.1 μl, 0.2 μl, 0.3 μl, 0.4 μl, 1 μl, 2 μl, 3 μl, 4 μl le 5 μl, ka ho latellana; NTC: taolo ntle le template Liphetho tsa liteko: Blood Direct PCR Kit e na le khanyetso e matla maling mme e ka matlafatsa disampole tsa mali ka lenane la ho kenya la 0.1-5 μl.
	Mehlala ea mali e tsoang ho batho, likhoto, likhoho le mefuta e meng e nang le kalafo e fapaneng e sebelisitsoe e le litempele. Blood Direct PCR Kit e ne e sebelisetsoa ho holisa PRNP (batho, 750 bp), Actin (rat, 200 bp), le β-Actin (Khoho, 1.0 kb). Sisteme ea PCR reaction e ne e le 20 μl, mme mali a 1 μl a sebelisitsoe e le template. M: TIANGEN Lesupa II. Liphetho tsa liteko: Blood Direct PCR Kit e ka sebelisoa mefuteng e mengata e fapaneng, 'me tlhahlobo e tobileng ea PCR e ka etsoa ka mehlala ea mali ea mefuta e fapaneng e nang le kalafo e fapaneng.

Q: Ha ho na lihlopha tsa matlafatso

Setšoantšo sa A-1

■ Template e na le litšila tsa protheine kapa li-inhibitors tsa Taq, jj. ——Tlhoekisa template ea DNA, tlosa litšila tsa protheine kapa u ntše template ea DNA ka lisebelisoa tsa tlhoekiso.

■ Tlatsetso ea template ha e felle ——Ho eketsa ka mokhoa o nepahetseng mocheso oa "denaturation" le ho lelefatsa nako ea denaturation.

■ Ho senyeha ha litempele ——Reka template hape.

Tlhahiso ea A-2

■ Boleng bo tlase ba li-primers ——Re kopanya hape selelekela.

■ Ho senyeha ha li-primer——Fana ka litlhahlobo tse phahameng tsa likhakanyo hore e be molumo o monyane bakeng sa ho li boloka. Qoba ho hatsela hangata le ho qhibiliha kapa nako e telele ea 4 ° C e bolokiloeng.

■ Moralo o sa nepahalang oa litlhahlobo (mohlala, bolelele ba lithuso ha boa lekana, botenya bo thehiloe lipakeng tsa lintho tsa pele, jj.

EA-3 Mg²⁺ho tsepamisa maikutlo

■ Mg²⁺ ho tsepamisa mohopolo ho tlase haholo ——Ho eketsa hantle Mg²⁺mahloriso: Lokisetsa Mg²⁺ mahloriso ka letoto la likarabelo ho tloha ho 1 mM ho isa ho 3 mM ka karohano ea 0.5 mM ho fumana Mg²⁺ mahloriso bakeng sa template le primer ka 'ngoe.

Mocheso oa ho koala oa A-4

■ Mocheso o phahameng oa ho koala o ama tlamo ea selelekela le template. Fokotsa mocheso o kenang 'me u ntlafatse boemo ka gradient ea 2 ° C.

Nako ea katoloso ea A-5

■ Nako e khuts'oane ea katoloso —- Eketsa nako ea katoloso.

P: Ho ba le tšoaetso ea bohata

Phenomena: Mehlala e mebe e bonts'a lihlopha tsa tatellano.

Tšilafalo ea A-1 ea PCR

■ Tšilafalo ea sefapano ea tatellano ea sepheo kapa lihlahisoa tsa ntlafatso ——Tlhokomelo hore o se ke oa tšela sampole e nang le tatellano ea sepheo sa sampuli e fosahetseng kapa oa e tšolla ka har'a tube ea centrifuge. Li-reagents kapa lisebelisoa li lokela ho etsoa mochini o ikemetseng ho felisa li-acid tsa nucleic, mme boteng ba tšilafalo bo lokela ho khethoa ka liteko tse mpe tsa taolo.

■ Tšilafalo ea li-reagent ——Qobella li-reagents ebe u li boloka ka mocheso o tlase.

A-2 Tona-khōlōr

■ Mg²⁺ ho tsepamisa mohopolo ho tlase haholo ——Ho eketsa hantle Mg²⁺ mahloriso: Lokisetsa Mg²⁺ mahloriso ka letoto la likarabelo ho tloha ho 1 mM ho isa ho 3 mM ka karohano ea 0.5 mM ho fumana Mg²⁺ mahloriso bakeng sa template le primer ka 'ngoe.

■ Moralo oa semmuso o sa nepahalang, 'me tatellano ea sepheo e na le thuto ea bongoli le tatellano e sa amaneng le sepheo. ——Litlhahlobo tsa pele-pele.

Q: Kholiso e sa tobang

Phenomena: Lihlopha tsa kholiso ea PCR ha li lumellane le boholo bo lebelletsoeng, ebang ke bo boholo kapa bo bonyenyane, kapa ka linako tse ling lihlopha tse ikhethileng tse matlafatsang le lihlopha tse sa ikhethileng tsa matlafatso lia etsahala.

Tlhahiso ea A-1

■ Lintlha tse qalang tsa semmuso

——Re-design ea khale.

■ Khakanyo ea semmuso e phahame haholo —— Ho eketsa mocheso oa denaturation hantle le ho lelefatsa nako ea denaturation.

Mofuta oa A-2 Mg²⁺ ho tsepamisa maikutlo

■ Mokha oa Mg²⁺ mahloriso a phahame haholo -— Fokotsa hantle mohopolo oa Mg2 +: Lokisetsa Mg²⁺ mahloriso ka letoto la likarabelo ho tloha ho 1 mM ho isa ho 3 mM ka karohano ea 0.5 mM ho fumana Mg²⁺ mahloriso bakeng sa template le primer ka 'ngoe.

A-3 Thermostable polymerase

■ Palo ea enzyme e fetelletseng —— Fokotsa palo ea enzyme ka nepo ka linako tse ling tsa 0.5 U.

Mocheso oa ho koala oa A-4

■ Mocheso o kenyang letsoho o tlase haholo —— Ho eketsa mocheso oa ho koala ka nepo kapa ho sebelisa mokhoa oa mekhahlelo e 'meli

Lipotoloho tsa A-5 PCR

■ Lipotoloho tse ngata tsa PCR ——Ho fokotsa palo ea lipotoloho tsa PCR.

P: Lihlopha tse hlabang kapa tse tlotsang

Tlhahiso ea A-1—— Boemo bo ikgethang ba maemo a mabe— - Iketsetse meralo ea mantlha, fetola boemo le bolelele ba semmuso ho ntlafatsa botumo ba eona; kapa ho etsa sehlaha sa PCR.

DNA ea A-2

——Thempleite ha e ea hloeka— - Hloekisa template kapa u ntše DNA e nang le lisebelisoa tsa tlhoekiso.

EA-3 Mg²⁺ ho tsepamisa maikutlo

——Mahl²⁺ ho tsepamisa mohopolo ho phahame haholo -— Fokotsa hantle Mg²⁺ mahloriso: Lokisetsa Mg²⁺ mahloriso ka letoto la likarabelo ho tloha ho 1 mM ho isa ho 3 mM ka karohano ea 0.5 mM ho fumana Mg²⁺ mahloriso bakeng sa template le primer ka 'ngoe.

E-4 dNTP

——Khokahano ea li-dNTP li holimo haholo —— Fokotsa mohopolo oa dNTP ka nepo

Mocheso oa Annealing oa 5

——Thempereichara e tlaase e kenang ——Ho eketsa hantle mocheso o tlatsetsang

Lipotoloho tsa A-6

—Kenale palo ea potoloho

P: Ke template ea bokae ea DNA e lokelang ho kenyelletsoa ho 50 μl PCR reaction system?

P: U ka holisa likaroloana tse telele joang?

Mohato oa pele ke ho khetha polymerase e loketseng. Kamehla Taq polymerase e ke ke ea bala liphoso ka lebaka la ho haelloa ke ts'ebetso ea 3'-5 'ea tokollo,' me ho se tšoane hantle ho tla fokotsa ts'ebetso ea likhechana. Ka hona, Taq polymerase e tloaelehileng e ke ke ea holisa hantle likaroloana tsa sepheo tse kholo ho feta 5 kb. Taq polymerase e nang le phetoho e khethehileng kapa botšepehi bo phahameng ba polymerase e lokela ho khethoa ho ntlafatsa katleho le ho fihlela litlhoko tsa sehlotšoana se selelele. Ntle le moo, kholiso ea likhechana tse telele e boetse e hloka phetoho e tsamaellanang ea moralo oa khale, nako ea denaturation, nako ea katoloso, pH ea buffer, jj. Hangata, li-primers tse nang le 18-24 bp li ka lebisa ho chai e ngata. Bakeng sa ho thibela tšenyo ea template, nako ea denaturation ho 94 ° C e lokela ho fokotsoa ho isa ho 30 sec kapa ka tlase ho potoloho, mme nako ea ho nyoloha mocheso ho ea ho 94 ° C pele amplification e lokela ho ba tlase ho 1 min. Ho feta moo, ho beha mocheso oa katoloso ka 68 ° C le ho rala nako ea katoloso ho latela sekhahla sa 1 kb / min ho ka netefatsa kholiso e sebetsang ea likhechana tse telele.

Q: U ka ntlafatsa ts'epahalo ea kholiso ea PCR joang?

Sekhahla sa phoso ea kholiso ea PCR se ka fokotsoa ka ho sebelisa li-polymerase tse fapaneng tsa DNA ka bots'epehi bo phahameng. Har'a li-polymerase tsohle tsa Taq DNA tse fumanehang ho fihlela joale, enzyme ea Pfu e na le sekhahla se tlase sa phoso le botšepehi bo phahameng ho fetisisa (bona tafole e hoketsoeng). Ntle le khetho ea enzyme, bafuputsi ba ka tsoela pele ho fokotsa sekhahla sa phetoho ea PCR ka ho ntlafatsa maemo a karabelo, ho kenyeletsoa le ho ntlafatsa sebopeho sa buffer, polasetiki ea thermostable polymerase le ho ntlafatsa palo ea potoloho ea PCR.

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