Kit ɗin PCR kai tsaye

Cat No: HCR2020A

Plant Direct PCR Kit ya dace da haɓaka ganyen tsire-tsire, iri, da sauransu, kuma ana iya amfani da shi don babban aikin nunawa na samfuran shuka waɗanda ba su ƙunshi polysaccharides da polyphenols ba.Ƙwaƙwalwar DNA polymerase kai tsaye dangane da juyin halitta da aka jagoranta yana da mafi girman juriya ga masu hana PCR a cikin tsire-tsire.A halin yanzu, yana kula da babban aikin haɓakawa, wanda ya dace da haɓaka gutsuttsuran DNA a cikin 5 kb.Ana iya amfani da buffer na musamman na Lysis A a cikin kit ɗin don lyse sabo ko daskararrun kyallen jikin shuka.Yana da sauƙin aiki kuma ana iya amfani da lysate azaman samfuri don haɓakawa ba tare da tsarkakewa ba.Tsarin yana ƙunshe da jami'ai masu kariya waɗanda ke ba da damar samfuran ɗanyen da za a haɓaka su yadda ya kamata bayan daskarewa da narke mai maimaitawa.2 × Plant Direct Master Mix kawai yana buƙatar ƙara ƙirar ƙira da samfura don aiwatar da haɓaka haɓakawa, ta haka rage ayyukan bututu da haɓaka abubuwan ganowa da sake haifar da sakamako.

Abubuwan da aka gyara

* Plant Direct Lysis Buffer B shine reagent na zaɓi, wanda ake amfani dashi don kawar da Plant Direct Lysis Buffer A don tsawaita lokacin ajiyar samfuran.Ana iya amfani da shi bisa ga ainihin halin da ake ciki.

Yanayin Ajiya

2 × Plant Direct Master Mix, adana a -30 ~ -15 ℃ kuma kauce wa daskarewa maimaituwa da narke;Plant Direct Lysis Buffer, adana a -30 ~ -15℃ ko 2 ~ 8℃.

Tsarin Gwaji

Samfuran Gudanarwa-Ganyen Shuka

Hanyar kai tsaye:Ana ba da shawarar yin amfani da ganyayyaki matasa.Yi amfani da buɗaɗɗen rami tare da ƙayyadadden diamita na 0.5 - 3 mm don samun ƙananan samfurori da kayan aiki, sa'an nan kuma ƙara samfurin kai tsaye zuwa tsarin PCR (ana bada shawarar tsarin 50 μl).Lura, tabbatar da samfurin yana cikin maganin PCR kuma ba akan bangon bututu ba.Idan ana amfani da PCR kai tsaye don haɓaka dogon gutsuttsura da samfurori masu rikitarwa, ta yin amfani da samfurin tare da ƙaramin diamita (0.5 - 1 mm) azaman samfuri na iya taimakawa wajen samun sakamako mai kyau.

Hanyar lysis nika:Ana ba da shawarar yin amfani da ganyayyaki matasa.Ɗauki ɗan ƙaramin ganye (kimanin 1 - 3 mm a diamita), sanya shi a cikin 20 μl Plant Direct Lysis Buffer Ab, kuma a niƙa shi gwargwadon iyawa (ana iya yin wannan mataki ta hanyar matse ganye tare da tip 100 μl pipette). don mash samfurin).Idan ana amfani da mafi girma na nama na ganye (ba su wuce 7 mm ba), ƙara ƙarar buffer dilution zuwa 50 μl.Bayan an yi ganyen ganye, maganin ya kamata ya bayyana kore.Bayan taƙaitaccen centrifugation, ƙara 1 μl na supernatant zuwa tsarin PCR azaman samfurin amsawa.

Hanyar thermal lysis:Ana ba da shawarar yin amfani da ganyayyaki matasa.Ɗauki ɗan ƙaramin ganye (kimanin 1 - 3 mm a diamita), sanya shi a cikin 20 μl Plant Direct Lysis Buffer A, kuma zafi shi a 95 ° C na 5 - 10 min.Za'a iya tsawaita lokacin lysis daidai ga ganye waɗanda ke da wahalar lyse (ba fiye da minti 20 ba).Idan ana amfani da mafi girma na nama na ganye (ba su wuce 7 mm ba), ƙara ƙarar buffer dilution zuwa 50 μl.Bayan dumama, centrifuge a taƙaice, kuma ƙara 1 μl na supernatant zuwa tsarin PCR azaman samfurin amsawa.

Samfuran Gudanarwa–Irin Shuka

Hanyar lysis nika:Yi amfani da ƙwanƙwasa don yanke tsaba tare da diamita na 5 mm, ƙara su zuwa 100 μl na Plant Direct Lysis Buffer A, sannan a niƙa samfurin tare da tip pipette ko wasu kayan aiki.Vortex a takaice kuma bari ya tsaya a dakin da zafin jiki na 3 - 5 min.Tabbatar cewa samfurin iri ya nutse a cikin ma'ajin dilution.Bayan taƙaitaccen centrifugation, ƙara 1 μl na supernatant zuwa tsarin PCR azaman samfurin amsawa.

Hanyar thermal lysis:Yi amfani da ƙwanƙwasa don yanke tsaba tare da diamita na 5 mm, ƙara su zuwa 100 μl na Plant Direct Lysis Buffer A, kuma zafi a 95 ° C na 5 - 10 min.Za'a iya tsawaita lokacin lysis daidai ga ganye waɗanda ke da wahalar lyse (ba fiye da 30 min ba).Bayan dumama, centrifuge a taƙaice, kuma ƙara 1 μl supernatant zuwa tsarin PCR azaman samfurin amsawa.

a.Hakanan za'a iya amfani da almakashi ko wasu kayan aikin don yanke samfuran girman da suka dace;idan an sake amfani da naushi ko almakashi, ya kamata a tsaftace su tare da maganin 2% sodium hypochlorite kafin kowane amfani don hana kamuwa da cuta tsakanin samfurori.

b.Tabbatar cewa Plant Direct Lysis Buffer ya narke sosai kafin amfani.Idan buffer yana da danko ko yana da hazo, ana iya dumama shi a 37 ℃ don narkewa gaba daya kafin amfani.

c.Za'a iya daidaita ƙarar samfuri a cikin tsarin amsawa daidai gwargwadon bambancin girman kayan shuka da ƙarar diluent.

Shuka Direct Lysis Buffer

Plant Direct Lysis Buffer A wanda ke ƙunshe a cikin wannan samfurin an inganta shi sosai don sakin kwayoyin halittar mafi yawan ƙwayoyin shuka kuma ya dace da adana ɗanyen tsire-tsire na ɗan lokaci a 4℃.Idan samfurin yana buƙatar adanawa na tsawon lokaci (misali, 1 - 2 watanni), ana bada shawara don canja wurin supernatant zuwa sabon bututun EP kuma adana shi a -20 ℃.Don adana samfuran da ƙarfi, ƙara daidai girman girman Plant Direct Lysis Buffer B zuwa babban abin da aka canjawa wuri, haɗa da kyau kuma adana a -20 ℃.Tsayayyen lokacin ajiya ya bambanta da samfuran shuka da jihohi.

Tsarin martani

a.Ya ƙunshi Mg²⁺a matakin ƙarshe na 2 mM.

b.Ana ba da shawarar yin amfani da ƙaddamarwa na ƙarshe na 0.4μM don kowane firamare.Yin amfani da firamare da yawa zai haifar da ƙara haɓakawa mara iyaka.

c.Ana iya daidaita adadin samfurin da aka yi amfani da shi bisa ga ainihin halin da ake ciki.Adadin da aka yi amfani da shi a cikin amsa guda ɗaya na samfurin lysed mai ɗanɗano za a iya daidaita shi tsakanin 2% - 20% na jimlar ƙarar amsawa.Yin amfani da samfurori da yawa na iya haifar da gazawar haɓakawa.

Shirin mayar da martani

a.Denaturation na farko (98 ℃, 5 min) yana haɓaka lysis na nama na shuka, yana sakin DNA na genomic wanda za'a iya amfani dashi don haɓaka PCR.Kada a rage lokaci ko rage zafin jiki.

b.Ana ba da shawarar saita shi daidai da ƙimar Tm na farko ko 2 ~ 4℃ sama da ƙimar Tm.Ƙwaƙwalwar DNA polymerase kai tsaye da aka yi amfani da ita a cikin wannan samfurin ya bambanta da na al'ada Taq DNA polymerase, kuma yana da buƙatu na musamman don yanayin zafi mai raɗaɗi;amfani da babban zafin jiki na iya rage ƙaƙƙarfan haɓakawa da kyau da haɓaka haɓakar haɓakawa.Don hadaddun samfura, ana iya samun ingantaccen haɓakawa ta hanyar daidaita yanayin zafi da tsawaita lokacin tsawo.

c.Idan tsawon samfurin haɓakawa shine ≤1 kb, an saita lokacin tsawo a 30 sec / kb;idan tsawon samfurin ƙarawa> 1 kb, an saita lokacin tsawo a 60 sec/kb.

d.Don hadaddun samfura ko samfurori tare da ƙananan haɓakar haɓakawa, ana iya ƙara adadin hawan keke daidai gwargwado zuwa zagayowar 40 -50.

Aikace-aikace

Ana amfani da shi don haɓaka kyallen takarda kai tsaye da kuma babban aikin nunawa na samfuran shuka waɗanda ba su ƙunshi polysaccharides da polyphenols ba.

Bayanan kula

Don bincike kawai amfani.Ba don amfani a cikin hanyoyin bincike ba.

1. Don haɓakar ɗanyen tsiro ko haɓakawa kai tsaye, ana ba da shawarar yin amfani da DNA mai tsaftataccen genomic azaman ingantaccen iko kafin fara gwajin don tabbatar da cewa tsarin, firam da ayyuka daidai ne.

2. Ƙarfafa DNA polymerase kai tsaye da aka yi amfani da shi a cikin wannan kit ɗin yana da aikin tantancewa mai ƙarfi.Idan TA cloning yana buƙatar yin, ana bada shawara don tsarkake DNA kafin ƙara adenine.

3. Jagorar Zane na Farko:

a.Ana ba da shawarar cewa tushe na ƙarshe a ƙarshen 3′ na farkon ya zama G ko C.

b.Ya kamata a guje wa rashin daidaituwa a jere a cikin tushe 8 na ƙarshe a ƙarshen 3′ na farkon.c.Guji tsarin gashin gashi a ƙarshen 3′ na farko.

d.Bambance-bambancen da ke cikin ƙimar Tm na gaba na gaba da na baya baya kamata ya zama fiye da 1 ℃ kuma ƙimar Tm yakamata a daidaita shi zuwa 60 ~ 72 ℃ (An ba da shawarar Premier 5 don ƙididdige ƙimar Tm).

e.Ƙarin ƙarin jeri na firamare waɗanda basu dace da samfuri ba, bai kamata a haɗa su ba yayin ƙididdige ƙimar Tm na farko.

f.Ana ba da shawarar cewa abun ciki na GC na farkon ya zama 40% -60%.

g.Rarraba gabaɗaya na A, G, C da T a cikin firamare ya kamata ya kasance gwargwadon yiwuwa.A guji amfani da yankuna masu babban abun ciki na GC ko AT.

h.Ka guji kasancewar ƙarin jerin tushe na 5 ko fiye ko dai a cikin na farko ko tsakanin firamare biyu kuma ka guje wa kasancewar ƙarin jeri na 3 ko fiye a ƙarshen 3′ na firamare biyu.

i.Yi amfani da aikin NCBI BLAST don bincika ƙayyadaddun ƙayyadaddun na'urar don hana haɓakawa na musamman.