2 × Taq Platinum PCR Mix
Ma'anar Ayyuka
Nau'in 1 (U) Taq Platinum DNA Polymerase aiki an bayyana shi azaman adadin enzyme da ake buƙata don haɗa 10 nmol deoxynucleotides cikin abubuwan da ba za su iya narkewa a 74 ° C a cikin mintin 30 ta amfani da salmon DNA mai aiki azaman samfuri/fitila.
Sarrafa Inganci
Tsarkakewa ta gano SDS-PAGE ya fi 99%; Ba a gano wani aiki na nuclease na waje ba; Kwayar halitta guda ɗaya a cikin ƙwayoyin halittar ɗan adam ana iya haɓaka ta yadda yakamata; Babu wani muhimmin canji na aiki idan aka adana shi a zafin jiki na ɗaki na mako guda.
Babban Siffofin Fasaha
Yana da aikin fitarwa na 5′-3 and da aikin 3′-5 ′, kuma amincinsa yana kusa da Pfu polymerase. Saurin fadada Taq Platinum Polymerase yana da sauri fiye da Pfu polymerase kuma ingancin faɗaɗa ya fi girma. Ana iya haɗa samfuran PCR kai tsaye zuwa ƙarshen m ko rufe tare da vector TA. Idan ana buƙatar inganta ingancin cloning, ana ba da shawarar yin tsarkakewa da farko kuma ƙara 3'-dA overhangs kafin cloning cikin TA vector.
-Aya-bututu Taq Platinum MasterMix (Takaddar Samfurin Babban Fasaha ta Ƙasa)
Ta Taq Platinum MasterMix ya inganta keɓancewa da ƙimar PCR kuma yana iya haɓaka samfuran hadaddun tare da babban abun ciki na GC, tsarin sakandare da makamantan su. Har zuwa kwafi 2 na samfuran da aka yi niyya za a iya haɓaka su, yana tabbatar da ingantattun sakamakon gwaji.
Formula Tsarin musamman na Taq Platinum MasterMix yana sa tsarin amsa duka ya tabbata sosai, kuma aikin ba zai shafar daskarewa-narkewa ko adana dogon lokaci a 4 ° C ba.
■ Tabbatacce da ingantaccen maganin PCR da aka riga aka shirya zai iya yin aikin cikin sauri da sauƙi, yana rage tsananin ƙarfin aiki da kuskuren samfuri. Hakanan ana haɓaka haɓakar PCR mai haɓakawa da haɓakawa a cikin cakuda, wanda ke rage buƙatun akan yanayin PCR.
■ Wannan samfurin yana da tsarin da ya ƙunshi fenti da fenti. Samfuran MasterMix mai launin fenti za a iya yin electrophoresed kai tsaye bayan PCR, ba tare da ƙara bulogin lodin ba.
Aikace -aikace
Zai iya maye gurbin Pfu polymerase don haɓaka samfuran aminci daga samfura masu rikitarwa kamar su kwayoyin halitta, kuma ya dace da aikace-aikace kamar cloning of genes expression, maye gurbin takamaiman rukunin yanar gizo da kuma nazarin polymorphism guda ɗaya na nucleotide (SNP), da sauransu.
Kariya a cikin ƙira Firayim Minista PCR:
Tsawon fitila yawanci shine 20-25 mer. Koyaya, lokacin yin PCR guntu mai tsayi, yakamata a ƙara girman matakin farko zuwa 30-35 mer.
■ Babu haɗin haɗin kai tsakanin firam ɗin biyu, musamman don tushen 3 na ƙarshe a ƙarshen ′.
Content Abubuwan GC yakamata su kasance 50-60%, kuma ku guji GC ko AT mai wadata. Domin yin ƙulli da samfuri mai ƙarfi, ku guji tsarin AT mai ƙima a ƙarshen 3 ′.
■ Kauce wa share fage don samar da tsarin sakandare.
■ Zaɓi firiji biyu tare da yanayin Tm kusa da juna.
Lissafin ƙimar Tm na Primers don PCR:
■ Lokacin da firam ɗin bai wuce 20 ba: Tm = 2 ° C × (A+T)+4 ° C × (G+C).
■ Lokacin da firam ɗin ya wuce 20 mer: Tm = 81.5+0.41 × (GC%)-600/L, inda L shine tsayin faramar.
■ Saita zafin zafin a (Tm-5) ° C.
Rahoton da aka ƙayyade na PCR
Za'a iya zaɓar madaidaicin ƙimar firam ɗin tsakanin 0.1 μM da 1.0 μM. Rashin ƙarancin firamare yana haifar da ƙarancin samfuran samfuran haɓakawa, yayin da ƙima mai yawa ya fi dacewa da haɓaka takamaiman. Yawancin lokaci, lokacin da adadin samfuran DNA ya yi yawa ko hadaddun samfuri na DNA (kamar DNA na ɗan adam) ana amfani da shi azaman samfuri, maida hankali na farko ya zama ƙasa. Lokacin da ake amfani da adadin samfur na DNA ƙarami ko sauƙi samfuri DNA (misali, plasmid DNA, da sauransu) azaman samfuri, maida hankali na farko ya zama mafi girma.
Duk samfuran ana iya keɓance su don ODM/OEM. Don cikakkun bayanai,don Allah danna Sabis na Musamman (ODM/OEM)
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Yi amfani da DNA na genomic azaman samfuri don haɓaka guntu 1 kb.Bayan halayen PCR, ɗauki 5 μl don gano electrophoresis. |
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.
Kayan samfuran
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