Igazi ngqo lePC Kit

Ukukhulisa ngokukhawuleza kohlobo olujolisiweyo kusetyenziswa ngokuthe ngqo igazi njengetemplate ngaphandle kokukhupha.

Le khithi yamkela imfuza yobuchwephesha ye-anti-inhibitor ye-DNA polymerase ngokufanelekileyo ukukhulisa imfuza yekopi enye kwi-genome yomntu. Inkqubo ye-buffer elungiselelwe kakuhle kule khithi inceda i-polymerase ukuba ixhathise ngamandla ukuthintela ii-PCR inhibitors, yiyo loo nto inokukhulisa ngokuthe ngqo i-DNA isebenzisa iiseli zegazi kunye nezicwangcisiweyo njengeetemplate. Le mveliso kulula ukuyisebenzisa kwaye ayifuni manyathelo anzima njengokucocwa kwe-DNA okanye isampulu yokunyanga kwangaphambili.
Le khithi ibonelelwe njenge-2 × MasterMix, kwaye impendulo inokwenziwa ngokudibanisa nje itemplate yegazi kunye nezinto zokuqala zokufumanisa. Ingasetyenziswa kwiiseli ezikhulisiweyo zezilwanyana ezifana nabantu, iigundane, iihagu, iinkomo kunye nezinye iintlobo, kunye negazi elitsha okanye eli-4 ℃ eligcinwe ngokupheleleyo, i-anticoagulant (i-EDTA, i-citrate, i-heparin), amahlwili egazi kunye namachaphaza egazi igcinwe kwi-Whatman 903 kunye ne-FTA Elute amakhadi entengiso.

Ikati. Hayi	Ubungakanani bokuPakisha
4992529	20 ×l × 100 rxn
4992530	20 ×l × 500 rxn

Thumela i-imeyile kuthi

Iinkcukacha Product

Umzekelo wovavanyo

FAQ

Iimpawu zemveliso

Iimbonakalo

Simple Elula kwaye iyakhawuleza: Ukwandiswa kwe-PCR kunokwenziwa ngokuthe ngqo kusetyenziswa igazi njengetemplate, ngaphandle kwesidingo samanyathelo adinisayo okulungiselela isampulu kunye nokukhupha i-DNA.
■ Ukucoceka okuphezulu: Ukutsiba isampulu yangaphambi konyango kunye namanyathelo okutsala i-DNA anokunceda ukuthintela ukungcoliseka kweesampulu.
■ Ukuhamba okuphezulu: Ukuchongwa kwePCR kweesampulu ezinkulu kunokwenziwa ngokudibanisa ikhithi kunye neeplati zePCR ezingama-96/384.
■ iyunivesi eyomeleleyo: Le khithi inokuwandisa ngokufanelekileyo amaqhekeza aphezulu e-GC okanye iziqwenga ezinesakhiwo esimbaxa sesibini, kwaye ubude bokwandisa bunokufikelela kwi-5 kb.
■ Ukuxhathisa ngamandla koxinzelelo: Le khithi inokusetyenziselwa iintlobo ngeentlobo kunye neesampulu zegazi ezigcinwe ngeendlela ezahlukeneyo.

Izicelo

Iimveliso zePCR zale khithi ziqulathe "A" kwisiphelo se-3', esinokusetyenziselwa ngokuthe ngqo ukwenza i-vector cloning. Le khithi inokusetyenziselwa ukukhulisa iziqwengana ze-genomic ye-DNA, uhlalutyo oluphezulu lwezinto eziphilayo kunye nohlalutyo lwe-genotyping (olufana nokuchongwa kwemfuza).

Zonke iimveliso zingenziwa ngokwezifiso kwi-ODM / OEM. Iinkcukacha,nceda ucofe iNkonzo eChongiweyo (i-ODM / i-OEM)

Egqithileyo I-TIANcombi DNA Lyse kunye ne-Det PCR Kit

Okulandelayo: Imouse Tissue ngqo PCR Kit

	Sebenzisa i-anticoagulation yabantu ye-EDTA njengetemplate, iijethi ezi-4 ezinemixholo eyahlukeneyo ye-GC yandiswa yiGazi ngqo lePC Kit. Inkqubo yokuphendula ye-PCR yayiyi-20 μl, kwaye igazi le-1 μl lalisetyenziswa njengetemplate. M: UMarki II weTIANGEN; 1: Ubungakanani beqhekeza 1090 bp, umxholo we-GC 68.1%; 2: Ubungakanani beqhekeza 1915 bp, umxholo we-GC 70.4%; 3: Ubungakanani beqhekeza 448 bp, umxholo we-GC 74.8%; 4: Ubungakanani beqhekeza 1527 bp, umxholo we-GC 61.5%. Iziphumo zovavanyo: Igazi elithe ngqo lePCR Kit linokukhulisa ngokufanelekileyo iziqwengana zeDNA kunye nomxholo we-GC kuluhlu lwama-61.5% -74.8%, iphakamisa ukuba iyakwazi ukukhulisa iziqwenga ze-GC eziphezulu.
	Sebenzisa i-anticoagulation yabantu ye-EDTA njengetemplate, iijethi ezi-5 ezinobude obahlukeneyo (i-ActB, i-Prp, i-DN1.0, i-Hn2.0 kunye ne-Hn4.0) ziye zandiswa yi-Blood Direct PCR Kit. Inkqubo yokuphendula ye-PCR yayiyi-20 μl, kwaye igazi le-1 μl lalisetyenziswa njengetemplate. M: UMarki II weTIANGEN; 1-3: 3 iisampulu zegazi ezahlukeneyo; I-NTC: Ulawulo ngaphandle kwee-primers. Iziphumo zovavanyo: Igazi elithe ngqo lePC PC Kit linokukhulisa iziqwenga ngobude obude bube zii-4 kb, iphakamisa ukuba iyakwazi ukukhulisa iziqwengana ezinde.
	Sebenzisa i-anticoagulation ye-EDTA yabantu njengetemplate, iGazi ngqo lePC PC Kit lalisetyenziselwa ukufumanisa i-PCR yeesampulu zegazi ezahlukeneyo. Inkqubo yokuphendula ye-PCR yayiyi-20 μl, kwaye igazi le-1 μl lalisetyenziswa njengetemplate. M: UMarki II weTIANGEN; 1-9: inani lokulayisha kwegazi yi-0.1 μl, 0.2 μl, 0.3 μl, 0.4 μl, 1 μl, 2 μl, 3 μl, 4 μl kunye 5 μl, ngokwahlukeneyo; I-NTC: ulawulo ngaphandle kwetemplate Iziphumo zovavanyo: Igazi elithe ngqo le-PCR Kit linokuxhathisa okunamandla kwigazi kwaye linokukhulisa iisampulu zegazi kuluhlu lokulayisha lwe-0.1-5 μl.
	Iisampulu zegazi ezivela kubantu, kumabuzi, kwinkuku nakwezinye iintlobo zonyango olwahlukeneyo zazisetyenziswa njengeetemplate. Igazi ngqo le-PCR Kit lalisetyenziselwa ukwandisa i-PRNP (yabantu, i-750 bp), i-Actin (i-rat, i-200 bp), kunye ne-β-Actin (Inkukhu, i-1.0 kb). Inkqubo yokuphendula ye-PCR yayiyi-20 μl, kwaye igazi le-1 μl lalisetyenziswa njengetemplate. M: ISIPHAKAMISO SEXESHA II. Iziphumo zovavanyo: Igazi elithe ngqo le-PCR Kit linokusetyenziswa kuluhlu olubanzi lweesampulu, kwaye ukubonwa ngokuthe ngqo kwePCR kunokwenziwa kwiisampulu zegazi ezivela kwiintlobo ezahlukeneyo zonyango ezahlukeneyo.

Q: Akukho zixhobo zokukhulisa

Itemplate ye-A-1

■ Itemplate iqulethe ukungcola kweprotein okanye i-Taq inhibitors, njl njl. —Purify ithemplate ye-DNA, susa ukungcola kweprotein okanye ukhuphe i-DNA yeseti kunye neekiti zokucoca.

■ Ukuchazwa kweetemplate akuphelelanga ——Ukunyusa ngokufanelekileyo ubushushu bexesha kunye nokwandisa ixesha lokwenza oko.

■ Ukuhla komgangatho wetemplate- Lungisa kwakhona itemplate.

I-A-2 Primer

■ Umgangatho ophantsi wee-primers -—Re-synthesize the primer.

■ ukuthotywa kwePrimer——Faka iziqwengana eziphezulu zoxinaniso zibe ngumthamo omncinci wolondolozo. Kunqande ukukhenkceza okuninzi kunye nokunyibilika okanye ixesha elide i-4 ° C ikhutshiwe.

■ Uyilo olungafanelekanga lwezinto zokuqala (umz.

I-A-3 Mg²⁺Uxinzelelo

■ Mg²⁺ uxinzelelo luphantsi kakhulu ——Ukunyusa ngokufanelekileyo uMg²⁺Uxinzelelo: Lungiselela iMg²⁺ uxinzelelo ngothotho lwempendulo ukusuka kwi-1 mM ukuya kwi-3 mM ngexeshana le-0.5 mM ukumisela eyona Mg²⁺ Uxinzelelo lwetemplate nganye kunye ne-primer.

Ubushushu be-A-4 bokuTshintsha

The Amaqondo obushushu aphezulu afakela umbane achaphazela ukubopha kwento yokuqala kunye netemplate. —Ukunciphisa ubushushu obongezelelekileyo kwaye wandise imeko ngegradient eyi-2 ° C.

Ixesha le-5 lokongezwa

■ Ixesha elifutshane lokwandiswa- Yandisa ixesha lolwandiso.

Umbuzo: Ubuxoki

Phenomena: Iisampulu ezingathandekiyo zikwabonisa ukulandelelana kwebhendi.

Ungcoliseko lwe-A-1 lwe-PCR

■ Ungcoliseko lomnqamlezo wolandelelwano ekujoliswe kulo okanye iimveliso zokukhulisa ——Ukunyamekela ukungahambi ngesampulu yesampulu enolandelelwano ekujoliswe kulo kwisampulu engeyiyo okanye uchithe ityhubhu yecentrifuge. Izinto ezenziwayo okanye izixhobo kufuneka zenziwe ngokuzenzekelayo ukuze kupheliswe iicicic acid ezikhoyo, kwaye ubukho bengcoliseko kufuneka bugqitywe ngovavanyo olubi lokulawula.

■ Ungcoliseko olungenanjongo —Faka iizincedisi uze uzigcine kubushushu obuphantsi.

Inkulumbuso ye-A-2r

■ Mg²⁺ uxinzelelo luphantsi kakhulu ——Ukunyusa ngokufanelekileyo uMg²⁺ Uxinzelelo: Lungiselela iMg²⁺ uxinzelelo ngothotho lwempendulo ukusuka kwi-1 mM ukuya kwi-3 mM ngexeshana le-0.5 mM ukumisela eyona Mg²⁺ Uxinzelelo lwetemplate nganye kunye ne-primer.

■ Uyilo olungafanelekanga lokuqala, kwaye ulandelelwano ekujoliswe kulo lunehomeology ngolandelelwano olungajoliswanga. -Ukuyila ngokutsha ii-primers.

Q: Ukwandiswa okungachazwanga

I-Phenomena: Iibhendi zokunyusa i-PCR azihambelani nobungakanani obulindelweyo, nokuba bukhulu okanye bincinci, okanye ngamanye amaxesha zombini izibophelelo ezithile kunye neebhendi zokunyusa ezingachazwanga.

I-A-1 Primer

■ Ukuchaneka kokukodwa kokuqala

-Ukuyila ngokutsha.

■ Uxinzelelo lwe-primer luphezulu kakhulu — -Ukunyusa ngokufanelekileyo ubushushu be-denaturation kunye nokwandisa ixesha le-denaturation.

I-A-2 Mg²⁺ Uxinzelelo

■ UMg²⁺ Ukugxininisa kuphezulu kakhulu — -Ukunciphisa ngokufanelekileyo i-Mg2 + yoxinaniso: Sebenzisa i-Mg²⁺ uxinzelelo ngothotho lwempendulo ukusuka kwi-1 mM ukuya kwi-3 mM ngexeshana le-0.5 mM ukumisela eyona Mg²⁺ Uxinzelelo lwetemplate nganye kunye ne-primer.

A-3 Polymerase enokunyangeka

■ Isixa se-enzyme esigqithisileyo —— Nciphisa isixa se-enzyme ngokufanelekileyo kumakhefu e-0.5 U.

Ubushushu be-A-4 bokuTshintsha

■ Iqondo lobushushu lokufakelwa liphantsi kakhulu ——Ukunyusa ngokufanelekileyo ubushushu obongezwe okanye sebenzisa indlela yokubamba enezigaba ezibini

Imijikelezo ye-A-5 ye-PCR

■ Imijikelo ye-PCR emininzi kakhulu —— Nciphisa inani lemijikelezo ye-PCR.

Q: Iipatchy okanye i-smear band

I-A-1 PrimerUkucaciswa kokubi -Yila kwakhona i-primer, tshintsha indawo kunye nobude be-primer ukongeza ukungangqinelani kwayo; okanye wenze i-PCR enendawo yokuhlala.

I-A-2 template yeDNA

—Ithemplate ayihlambulukanga -Hlambulula itemplate okanye ukhuphe i-DNA eneekiti zokucoca.

I-A-3 Mg²⁺ Uxinzelelo

—Mg²⁺ ingxinano iphezulu kakhulu -—Ukunciphisa ngokufanelekileyo uMg²⁺ Uxinzelelo: Lungiselela iMg²⁺ uxinzelelo ngothotho lwempendulo ukusuka kwi-1 mM ukuya kwi-3 mM ngexeshana le-0.5 mM ukumisela eyona Mg²⁺ Uxinzelelo lwetemplate nganye kunye ne-primer.

I-4-dNTP

—— Uxinaniso lwe-dNTPs luphezulu kakhulu —— Nciphisa uxinaniso lwe-dNTP ngokufanelekileyo

Ubushushu be-5-Annealing

—— Amaqondo obushushu aphantsi athathelwa phantsi ——Ukunyusa ngokufanelekileyo iqondo lobushushu elihlanganisiweyo

Imijikelo ye-A-6

—Imijikelo emininzi ——Yandisa inani lomjikelo

Q: Yimalini itemplate yeDNA ekufuneka ifakwe kwi-50 μl PCR reaction system?

Umbuzo: Ungawandisa njani amaqhekeza amade?

Isinyathelo sokuqala kukukhetha i-polymerase efanelekileyo. Rhoqo Taq polymerase ayinakuphinda ifundwe ngenxa yokunqongophala kwemisebenzi ye-3'-5 'exonuclease, kwaye ukungangqinelani kuya kunciphisa kakhulu ukwandiswa kweziqwengana. Ke ngoko, i-Taq polymerase eqhelekileyo ayinako ukukhulisa ngokufanelekileyo iziqwengana ekujoliswe kuzo ezinkulu kune-5 kb. I-Taq polymerase enenguqulelo ekhethekileyo okanye enye i-high fidelity polymerase kufuneka ikhethwe ukuphucula ulwandiso kunye nokuhlangabezana neemfuno zokukhulisa iziqwengana ezinde. Ukongeza, ukukhula kwamaqhekeza amade kukwafuna uhlengahlengiso oluhambelanayo loyilo lokuqala, ixesha lokubonisa, ixesha lokwandiswa, i-pH yesixhobo, njl. Njl. Ukuthintela ukonakala kwetemplate, ixesha lokudlulisa kwi-94 ° C kufuneka licuthwe liye kwi-30 sec okanye ngaphantsi kumjikelo ngamnye, kwaye ixesha lokunyuka kobushushu liye kwi-94 ° C ngaphambi kokukhulisa kufuneka kube ngaphantsi komzuzu omnye. Ngapha koko, ukuseta ubushushu obandisiweyo malunga ne-68 ° C kunye nokuyila ixesha lolwandiso ngokwenqanaba le-1 kb / min kunokuqinisekisa ukukhulisa ngokufanelekileyo iziqwengana ezinde.

Q: Ungakuphucula njani ukunyaniseka kokukhulisa i-PCR?

Ixabiso lempazamo zokukhulisa i-PCR linokuncitshiswa ngokusebenzisa iipolymerase zeDNA ezahlukeneyo ngokuthembeka okuphezulu. Kuzo zonke i-Taq DNA polymerases ezifumanekayo ukuza kuthi ga ngoku, iPfu enzyme ineyona mpazamo iphantsi kunye nokunyaniseka okuphezulu (jonga itafile eqhotyoshelweyo). Ukongeza kukhetho lwe-enzyme, abaphandi banokuqhubeka nokunciphisa inqanaba lokutshintsha kwe-PCR ngokwandisa iimeko zokuphendula, kubandakanya nokwenza ukwakheka kwesixokelelwano, uxinzelelo lwe-polymerase enokunyanga kunye nokwenza nombolo yomjikelo wePCR.

Bhala umyalezo wakho apha uze uyithumele kuthi