Kit ɗin PCR na jini kai tsaye

Saurin faɗaɗa ƙwayar halittar da ake nufi kai tsaye ta amfani da jini azaman samfuri ba tare da cirewa ba.

Wannan kit ɗin yana ɗaukar ƙirar ƙirar rigakafin ƙwayar cuta ta DNA polymerase don haɓaka ingantattun kwayoyin halitta guda ɗaya a cikin tsarin ɗan adam. Ingantaccen tsarin buffata a cikin wannan kit ɗin yana taimaka wa polymerase ya yi tsayayya sosai da hana masu hana PCR, don haka yana iya haɓaka DNA kai tsaye ta amfani da jini da ƙwayoyin al'ada azaman samfura. Wannan samfur ɗin yana da sauƙin aiki kuma baya buƙatar matakai masu rikitarwa kamar tsarkakewar DNA ko samfuran samfura.
An ba da wannan kit ɗin azaman 2 × MasterMix, kuma za a iya aiwatar da martani ta hanyar ƙara samfuran jini da madaidaitan abubuwan ganowa. Ana iya amfani da shi akan ƙwayoyin al'adun dabbobi masu shayarwa kamar mutane, mice, aladu, shanu da sauran nau'ikan, kazalika da sabo ko 4 ℃ cryopreserved blood, anticoagulant (EDTA, citrate, heparin), daskararren jini da bushewar tabo. adana akan katunan kasuwanci na Whatman 903 da FTA Elute.

Cat. A'a	Girman shiryawa
4992529	20 µl × 100 rxn
4992530	20 µl × 500 rxn

Aika mana imel

Bayanin samfur

Misalin Gwaji

Tambayoyi

Alamar samfur

Siffofin

■ Mai sauƙi da sauri: ana iya aiwatar da haɓaka PCR kai tsaye ta amfani da jini azaman samfuri, ba tare da buƙatar matakai masu wahala na shirye -shiryen samfur da haɓakar DNA ba.
Urity Tsarkin tsarki: Tsallake samfurin riga-kafi da matakan hakar DNA na iya taimakawa don gujewa gurɓata samfuran.
■ Babban kayan aiki: Ana iya yin gwajin PCR don samfuran manyan sikeli ta hanyar haɗa kit ɗin tare da faranti PCR 96/384.
■ Ƙarfafawa ta duniya: Wannan kit ɗin na iya haɓaka manyan gutsuttsuran GC ko gutsuttsura tare da hadaddun tsarin sakandare, kuma tsayin ƙaramin zai iya kaiwa 5 kb.
Resistance Ƙarfin damuwa mai ƙarfi: Ana iya amfani da wannan kit ɗin don nau'ikan daban -daban da samfuran jini da aka adana ta hanyoyi daban -daban.

Aikace -aikace

Abubuwan PCR na wannan kit ɗin sun ƙunshi "A" a ƙarshen 3′-ƙarshen, wanda za'a iya amfani dashi kai tsaye don cloning vector TA. Za'a iya amfani da wannan kit ɗin don haɓaka gutsutsuren DNA na genomic, bincike mai zurfi na ƙwayoyin cuta da bincike na genotyping (kamar gano asalin halitta).

Duk samfuran ana iya keɓance su don ODM/OEM. Don cikakkun bayanai,don Allah danna Sabis na Musamman (ODM/OEM)

Na baya: TIANcombi DNA Lyse & Det PCR Kit

Na gaba: Mouse Tissue Direct PCR Kit

	Yin amfani da maganin kashe kumburin EDTA na mutum azaman samfuri, kwayoyin halittar 4 tare da abubuwan GC daban -daban sun haɓaka ta Kit ɗin Kai tsaye PCR. Tsarin amsawar PCR shine 20 μl, kuma an yi amfani da 1 bloodl jini azaman samfuri. M: Alamar TIANGEN II; 1: Girman guntu 1090 bp, abun cikin GC 68.1%; 2: Girman guntu 1915 bp, abun cikin GC 70.4%; 3: Girman guntu 448 bp, abun cikin GC 74.8%; 4: Girman guntu 1527 bp, abun cikin GC 61.5%. Sakamakon gwaji: Kit ɗin PCR na jini kai tsaye yana iya haɓaka gutsutsuren DNA yadda yakamata tare da abun cikin GC a cikin kewayon 61.5%-74.8%, yana ba da shawarar yana da ikon haɓaka gutsattsarin GC.
	Yin amfani da maganin kashe kumburin EDTA na mutum azaman samfuri, kwayoyin halittu 5 masu tsayin tsayi daban -daban (ActB, Prp, DN1.0, Hn2.0 da Hn4.0) sun haɓaka ta Kit ɗin PCR na Jini. Tsarin amsawar PCR shine 20 μl, kuma an yi amfani da 1 bloodl jini azaman samfuri. M: Alamar TIANGEN II; 1-3: 3 samfuran jini daban-daban; NTC: sarrafawa ba tare da firimiya ba. Sakamakon gwaji: Kit ɗin PCR na Kai tsaye na jini yana iya haɓaka gutsutsuren tare da tsawon har zuwa 4 kb, yana ba da shawarar yana da ikon haɓaka guntun guntun.
	Ta amfani da maganin kashe kumburin EDTA na mutum azaman samfuri, An yi amfani da Kit ɗin PCR na Jini don gano PCR na samfuran jini daban -daban. Tsarin amsawar PCR shine 20 μl, kuma an yi amfani da 1 bloodl jini azaman samfuri. M: Alamar TIANGEN II; 1-9: adadin lodin jini shine 0.1 μl, 0.2 μl, 0.3 μl, 0.4 μl, 1 μl, 2 μl, 3 μl, 4 μl da 5 μl, bi da bi; NTC: sarrafawa ba tare da samfuri ba Sakamakon gwaji: Kit ɗin PCR na Kai tsaye yana da juriya mai ƙarfi ga jini kuma yana iya haɓaka samfuran jini tare da kewayon ɗaukar nauyin 0.1-5 μl.
	Anyi amfani da samfuran jini daga ɗan adam, bera, kaji da sauran nau'ikan da ke da magunguna daban -daban azaman samfura. Anyi amfani da Kit ɗin PCR na jini don haɓaka PRNP (ɗan adam, 750 bp), Actin (bera, 200 bp), da β-Actin (Chicken, 1.0 kb). Tsarin amsawar PCR shine 20 μl, kuma an yi amfani da 1 bloodl jini azaman samfuri. M: TIANGEN Alamar II. Sakamakon gwaji: Za a iya amfani da Kit ɗin PCR na jini kai tsaye akan samfura masu yawa, kuma ana iya yin gwajin PCR kai tsaye akan samfuran jini daga nau'ikan daban -daban tare da jiyya daban -daban.

Tambaya: Babu makaɗan ƙarawa

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.

Tambaya: Tabbatacce

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.

Tambaya: Ƙarfafawa ba takamaimai ba

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.

Tambaya: Ƙungiyoyi masu ƙyalli ko shafa

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

Tambaya: Nawa samfuri nawa ne ya kamata a ƙara a cikin tsarin amsawa na 50 μl PCR?

Tambaya: Yadda za a fadada dogayen gutsutsuren?

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.

Tambaya: Yadda za a inganta amincin amincin PCR?

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.

Rubuta saƙonka a nan ka aika mana