2 Mix Haɗin Pfu PCR

Samfurin A-1

Template Samfurin yana ɗauke da ƙazamar furotin ko masu hana Taq, da dai sauransu ——- Tsarkake samfurin DNA, cire ƙazantar furotin ko cire samfuri na DNA tare da kayan tsarkakewa.

■ Ƙaddamar da samfuri bai cika ba ——Ya dace a ƙara yawan zazzabin denaturation da tsawaita lokacin denaturation.

Deg ƙasƙantar da samfuri ——Ka sake shirya samfuri.

Bayanan Bayani na A-2

Quality Kyakkyawan ingancin fitila — —Re-synthesize primer.

Deg ƙasƙantar da kai na farko — —Aliquot babban firam ɗin taro zuwa ƙaramin ƙara don adanawa. Guji daskarewa da narkewa da yawa ko adanawa na 4 ° C na dogon lokaci.

Design Tsararrun ƙirar firamari (misali tsawon firamare bai isa ba, dimer da aka kafa tsakanin firam ɗin, da dai sauransu) -Rirar ƙira (ku guji samuwar dimer primer da tsarin sakandare)

A-3 MG²⁺maida hankali

■ Mg²⁺ maida hankali yayi ƙasa sosai ——Yin haɓaka Mg daidai²⁺ maida hankali: Inganta Mg²⁺ maida hankali ta hanyar jerin halayen daga 1 mM zuwa 3 mM tare da tazara na 0.5 mM don ƙayyade mafi kyawun Mg²⁺ maida hankali ga kowane samfuri da share fage.

A-4 Ƙara yawan zafin jiki

Zazzabi mai ƙima yana shafar ɗaurin firamare da samfuri. —— Rage zafin zafin zafin jiki da inganta yanayin tare da ɗimbin digiri na 2 ° C.

A-5 Tsawaita lokacin

■ Gajerun lokacin ƙara —— Ƙara lokacin ƙarawa.

Phenomena: Samfuran marasa kyau suma suna nuna makasudin jerin makasudin.

A-1 Gurɓatar PCR

Tsallake gurɓataccen jerin abubuwan da aka yi niyya ko samfuran ƙarfafawa ——Ayi hankali kada a ɗora samfurin da ke ɗauke da jerin manufa a cikin samfurin mara kyau ko zubar da su daga bututun ƙarfe. Yakamata reagents ko kayan aiki suyi aiki da kan su don kawar da acid nucleic da ke akwai, kuma yakamata a ƙaddara wanzuwar gurɓatarwa ta hanyar gwajin sarrafawa mara kyau.

Contamin Gurɓatawa mai gurɓatawa ——Aliquot reagents da adanawa a ƙaramin zafin jiki.

A-2 Firayim Ministar

■ Mg²⁺ maida hankali yayi ƙasa sosai ——Yin haɓaka Mg daidai²⁺ maida hankali: Inganta Mg²⁺ maida hankali ta hanyar jerin halayen daga 1 mM zuwa 3 mM tare da tazara na 0.5 mM don ƙayyade mafi kyawun Mg²⁺ maida hankali ga kowane samfuri da share fage.

Tsarin ƙirar da bai dace ba, kuma jerin maƙasudin yana da homology tare da jerin marasa manufa. ——Re-design primers.

Phenomena: Ƙungiyoyin haɓaka PCR ba su dace da girman da ake tsammani ba, ko babba ko ƙarami, ko kuma wani lokacin duka takamaiman ƙungiyoyin haɓakawa da ƙamus ɗin ba na musamman ba.

Farashin A-1

Ƙididdiga ta musamman

——Re-design primer.

Concentration Mahimmancin taro ya yi yawa sosai ——Ya ƙara haɓaka zafin denaturation da tsawaita lokacin denaturation.

A-2 MG²⁺ maida hankali

M Mg²⁺ maida hankali ya yi yawa sosai ——Ya dace a rage maida hankali Mg2+: Inganta Mg²⁺ maida hankali ta hanyar jerin halayen daga 1 mM zuwa 3 mM tare da tazara na 0.5 mM don ƙayyade mafi kyawun Mg²⁺ maida hankali ga kowane samfuri da share fage.

A-3 Polymerase mai ƙarfi

Yawan adadin enzyme mai yawa ——Rage adadin enzyme yadda yakamata a tsakanin 0.5 U.

A-4 Ƙara yawan zafin jiki

Temperature Zazzabin ƙoshin ya yi ƙasa da yawa ——Ya dace a ƙara yawan zafin zafin ɗin ko a yi amfani da hanyar ƙara matakin biyu

A-5 PCR hawan keke

Cy Yawan hawan PCR da yawa —— Rage yawan hawan PCR.

Farashin A-1—— Bayanai marasa kyau ——Rayyana ƙirar fitila, canza matsayi da tsawon fitilar don haɓaka takamaiman sa; ko yin PCR da aka ƙera.

A-2 Template DNA

—— Samfurin ba shi da tsarki ——Ka tsarkake samfur ko cire DNA tare da kayan tsarkakewa.

A-3 MG²⁺ maida hankali

——Mg²⁺ maida hankali yayi yawa ——Ya rage Mg daidai²⁺ maida hankali: Inganta Mg²⁺ maida hankali ta hanyar jerin halayen daga 1 mM zuwa 3 mM tare da tazara na 0.5 mM don ƙayyade mafi kyawun Mg²⁺ maida hankali ga kowane samfuri da share fage.

A-4 dNTP

——Tinganin dNTPs sun yi yawa ——Rage taro na dNTP yadda yakamata

A-5 Ƙara yawan zafin jiki

—— Ƙaramin zafin zafin ƙanƙara mai zafi ——Ya dace a ƙara yawan zafin zafin

Hanyoyin A-6

——Yawan hawan keke da yawa ——Ya inganta lambar zagayowar

Mataki na farko shine zaɓi polymerase da ya dace. Taq polymerase na yau da kullun ba zai iya sake karantawa ba saboda rashin aikin fitowar 3'-5 ', kuma rashin daidaituwa zai rage ƙimar fadada guntu. Sabili da haka, polymerase na Taq na yau da kullun ba zai iya haɓaka gutsattsarin da ya fi girma fiye da 5 kb ba. Taq polymerase tare da gyare -gyare na musamman ko wani babban aminci polymerase yakamata a zaɓi don haɓaka ingantaccen haɓakawa da saduwa da buƙatun ƙaramin guntu. Bugu da ƙari, ƙaramin guntun gutsutsuren kuma yana buƙatar daidaita daidaiton ƙirar fitila, lokacin denaturation, lokacin faɗaɗawa, pH mai ɓoyewa, da sauransu. Don hana lalacewar samfuri, lokacin denaturation a 94 ° C yakamata a rage zuwa 30 sec ko ƙasa da kowane zagayowar, kuma lokacin haɓaka zafin jiki zuwa 94 ° C kafin haɓaka ya zama ƙasa da 1 min. Haka kuma, saita zafin zafin a kusan 68 ° C da kuma tsara lokacin tsawaita gwargwadon ƙimar 1 kb/min na iya tabbatar da ingantaccen ingantaccen guntun gutsutsuren.

Ana iya rage ƙimar kuskuren haɓaka PCR ta amfani da polymerases DNA daban -daban tare da babban aminci. Daga cikin duk polymerases na Taq DNA da aka samo zuwa yanzu, enfuz enzyme yana da mafi ƙarancin ƙimar kuskure da mafi aminci (duba tebur da aka makala). Baya ga zaɓin enzyme, masu bincike na iya ƙara rage ƙimar maye gurbi ta PCR ta hanyar inganta yanayin amsawa, gami da haɓaka abun da ke ciki, maida hankali na polymerase mai ɗorewa da haɓaka lambar sake zagayowar PCR.