2 × Umxube wePfu we-PCR

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.

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.

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.

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

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.

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.