Tshala ikhithi ye-PCR eqondile
Inombolo yekati: HCR2020A
I-Plant Direct PCR Kit ilungele ukukhuliswa okuqondile kwamaqabunga ezitshalo, imbewu, njll., futhi ingasetshenziselwa ukuhlolwa okuphezulu kwamasampula ezitshalo angaqukethe ama-polysaccharides nama-polyphenols.I-DNA polymerase ye-amplification eqondile esekelwe ekuziphendukeleni kwemvelo okuqondisiwe inokubekezelela okuphakeme kuma-PCR inhibitors ezitshalweni.Khonamanjalo, igcina ukusebenza kokukhulisa okuphezulu, okulungele ukukhuliswa kwezingcezu ze-DNA phakathi kuka-5 kb.I-Lysis buffer A ehlukile kukhithi ingasetshenziswa ukuhlanganisa izicubu zezitshalo ezintsha noma eziqandisiwe.Kulula ukusebenza futhi i-lysate ingasetshenziswa njengesifanekiso sokukhulisa ngaphandle kokuhlanzwa.Uhlelo luqukethe ama-agent avikelayo avumela amasampula aluhlaza ukuthi akhuliswe ngempumelelo ngemva kokuqhwaza okuphindaphindiwe nokuncibilika.2 × I-Plant Direct Master Mix idinga kuphela ukwengeza iziqalo nezifanekiso ukuze yenze ukusabela kokukhulisa, ngaleyo ndlela kuncishiswe ukusebenza kwamapayipi nokuthuthukisa ukutholakala kokuphuma kanye nokuphindaphindeka kwemiphumela.
Izingxenye
| Izingxenye | 50 RXNS | 200 RXNS |
| 2 × Plant Direct Master Mix | 1.25 ml | 4×1.25 ml |
| Plant Direct Lysis Buffer A | 5 ml | 20 ml |
| Plant Direct Lysis Buffer B* | 5 ml | 20 ml |
*I-Plant Direct Lysis Buffer B iyi-reagent ongayikhetha, esetshenziselwa ukwenza i-Plant Direct Lysis Buffer A ingabi namandla ukuze kunwetshwe isikhathi sokugcina samasampuli.Ingasetshenziswa ngokuvumelana nesimo sangempela.
Izimo Zokugcina
2 × Plant Direct Master Mix, gcina ku--30 ~ -15℃ futhi ugweme ukuqhwa okuphindaphindiwe nokuncibilika;Plant Direct Lysis Buffer, gcina kokuthi -30 ~ -15℃ noma 2 ~ 8℃.
Inqubo Yokuhlola
Ukucutshungulwa kwesampula-Iqabunga Lesitshalo
Indlela eqondile:Kunconywa ukusebenzisa amaqabunga amancane.Sebenzisa i-punch yembobo enobubanzi obungu-0.5 - 3 mm ukuze uthole isampula encane neyunifomu, bese wengeza ngokuqondile isampula ohlelweni lwe-PCR (kunconywa uhlelo lwe-50 μl).Qaphela, qiniseka ukuthi isampula ikusixazululo se-PCR hhayi ngokumelene nodonga lweshubhu.Uma i-PCR eqondile isetshenziselwa ukukhulisa izingcezu ezinde namasampuli ayinkimbinkimbi, ukusebenzisa isampula enobubanzi obuncane (0.5 – 1 mm) njengesifanekiso kungasiza ekutholeni imiphumela engcono.
Indlela ye-lysis yokugaya:Kunconywa ukusebenzisa amaqabunga amancane.Thatha iqabunga elincane (elingaba ngu-1 – 3 mm ububanzi), ulibeke ku-20 μl Plant Direct Lysis Buffer Ab, bese uligaya ngangokunokwenzeka (lesi sinyathelo singenziwa ngokukhama iqabunga ngethiphu ye-pipette engu-100 μl. ukuhlanganisa isampula).Uma kusetshenziswa imiqulu emikhulu yamathishu amaqabunga (ungadluli ku-7 mm), khulisa umthamo we-dilution buffer ube ngu-50 μl.Ngemuva kokuthi amaqabunga aphansi, isisombululo kufanele sibonakale siluhlaza.Ngemva kwe-centrifugation kafushane, engeza u-1 μl we-supernatant ohlelweni lwe-PCR njengesifanekiso sokusabela .
Indlela ye-Thermal lysis:Kunconywa ukusebenzisa amaqabunga amancane.Thatha iqabunga elincane (elingaba ngu-1 - 3 mm ububanzi), ulibeke ku-20 μl I-Plant Direct Lysis Buffer A, bese uyishisa ku-95 ° C imizuzu engu-5 - 10.Isikhathi se-lysis singanwetshwa ngokufanelekile kumaqabunga okunzima ukuwahlanza (akukho ngaphezu kwemizuzu engama-20).Uma kusetshenziswa imiqulu emikhulu yamathishu amaqabunga (ungadluli ku-7 mm), khulisa umthamo we-dilution buffer ube ngu-50 μl.Ngemva kokufudumeza, beka i-centrifuge kafushane, bese wengeza u-1 μl we-supernatant ohlelweni lwe-PCR njengesifanekiso sokusabela.
Ukucutshungulwa kwesampula– Tshala Imbewu
Indlela ye-lysis yokugaya:Sebenzisa i-scalpel ukuze usike imbewu enobubanzi obungu-5 mm, uyingeze ku-100 μl we-Plant Direct Lysis Buffer A, bese ugaya isampula ngethiphu ye-pipette noma amanye amathuluzi.Vortex kafushane futhi ake ume ekamelweni lokushisa 3 - 5 min.Qiniseka ukuthi isampula lembewu licwile ku-dilution buffer.Ngemva kwe-centrifugation kafushane, engeza u-1 μl we-supernatant ohlelweni lwe-PCR njengesifanekiso sokusabela.
Indlela ye-Thermal lysis:Sebenzisa i-scalpel ukusika imbewu enobubanzi obungu-5 mm, yengeze ku-100 μl we-Plant Direct Lysis Buffer A, bese ushisa ku-95°C imizuzu engu-5 - 10.Isikhathi se-lysis singanwetshwa ngokufanelekile kumaqabunga okunzima ukuwahlanza (akukho ngaphezu kwemizuzu engama-30).Ngemva kokufudumeza, beka i-centrifuge kafushane, bese wengeza i-1 μl supernatant ohlelweni lwe-PCR njengesifanekiso sokusabela.
a.Izikele noma amanye amathuluzi nawo angasetshenziswa ukusika amasampula osayizi ofanele;uma isibhakela noma isikelo siphinda sisetshenziswa, kufanele sihlanzwe ngesisombululo se-sodium hypochlorite esingu-2% ngaphambi kokusetshenziswa ngakunye ukuze kuvinjelwe ukungcoliswa phakathi kwamasampuli.
b.Qinisekisa ukuthi i-Plant Direct Lysis Buffer incibilike ngokuphelele ngaphambi kokusetshenziswa.Uma isigcinalwazi sine-viscous noma sinezimvula, singashiswa ku-37℃ ukuze siyincibilike ngokuphelele ngaphambi kokusetshenziswa.
c.Umthamo wesifanekiso ohlelweni lokusabela ungalungiswa ngokufanele ngokuhambisana nomehluko wevolumu yezinto zezitshalo kanye ne-diluent eyengeziwe.
Plant Direct Lysis Buffer
I-Plant Direct Lysis Buffer A equkethwe kulo mkhiqizo yenziwe ngokugcwele ukuze ikhulule i-genome yezicubu eziningi zezitshalo futhi ifanele ukugcinwa kwezitshalo ezingahluziwe zesikhashana ku-4℃.Uma isampula idinga ukugcinwa isikhathi eside (isb, izinyanga ezingu-1 – 2), kutuswa ukuthi kudluliselwe i-supernatant kushubhu ye-EP entsha bese uyigcina ku--20℃.Ukuze ugcine amasampuli azinze kakhudlwana, engeza ivolumu elinganayo ye-Plant Direct Lysis Buffer B kumandla amakhulu adlulisiwe, hlanganisa kahle bese ugcine ku -20℃.Isikhathi sokugcina esizinzile siyahlukahluka kuye ngamasampula ezitshalo nezimo.
Isistimu yokusabela
| ddH2O | Ku-20.0 µl | Kufika ku-50.0µl |
| 2 × Plant Direct Master Mixa | 10.0µl | 25.0µ |
| I-Primer 1 (10 µM) | 0.8µl | 2.0µl |
| I-Primer 2 (10 µM)b | 0.8µl | 2.0µl |
| Isampula yeqabunga lezitshalo/isampula engahluziwe(Bheka Ukucubungula Isampula) | 0.5 – 3 mm leaf disc/x µl | 0.5 – 3 mm leaf disc/x µl |
a.Iqukethe i-Mg2+ekuhlanganiseni kokugcina okungu-2 mM.
b.Kunconywa ukusebenzisa ukuhlushwa kokugcina kwe-0.4μM ku-primer ngayinye.Ukusetshenziswa ngokweqile kwama-primer kuzoholela ekwandeni kokukhulisa okungaqondile.
c.Inani lesampula elisetshenzisiwe lingalungiswa ngokuya ngesimo sangempela.Inani elisetshenziswe ekuphenduleni okukodwa kwesampula ye-lysed engahluziwe ingalungiswa phakathi kuka-2% - 20% wesamba sevolumu yokusabela.Ukusebenzisa amasampula amaningi kakhulu kungabangela ukwehluleka kokukhulisa.
Uhlelo Lokusabela
| Izinyathelo | Izinga lokushisa | Isikhathi |
| I-Denaturation Yokuqala | 98℃ | 5 imiz |
| I-Denaturation | 95℃ | 10 isekhondi |
| Anealing | 58 ~ 72℃ | 15 isekhondi |
| Isandiso | 72℃ | 30 isekhondi |
| Isandiso Sokugcina | 72℃ | 5 imiz |
a.I-Initial Denaturation (98℃, 5 min) ikhuthaza ukuguqulwa kwezicubu zezitshalo, ikhiphe i-genomic DNA engasetshenziselwa ukukhulisa i-PCR.Ungasifinyezi isikhathi noma wehlise izinga lokushisa.
b.Kunconywa ukuthi uyimise ilingane nenani lokuqala le-Tm noma ngo-2 ~ 4℃ ngaphezu kwevelu ye-Tm.I-DNA polymerase ye-amplification eqondile esetshenziswe kulo mkhiqizo ihlukile kwejwayelekile ye-Taq DNA polymerase, futhi inezidingo ezikhethekile ze-reaction annealing temperature; ukusetshenziswa kwezinga lokushisa eliphezulu le-annealing kunganciphisa ngempumelelo ukukhulisa okungacacisiwe futhi kuthuthukise ukusebenza kahle kokukhulisa.Ezifanekiso eziyinkimbinkimbi, ukukhulisa okusebenzayo kungafinyelelwa ngokulungisa izinga lokushisa le-anneal nokwandisa isikhathi sokunwetshwa.
c.Uma ubude bomkhiqizo wokukhulisa umsindo bungu-≤1 kb, isikhathi sokunwetshwa sisethwa ku-30 sec/kb;uma ubude bomkhiqizo wokukhulisa i->1 kb, isikhathi sokunwetshwa sisethwe ku-60 sec/kb.
d.Kumasampuli ayinkimbinkimbi noma amasampuli anesivuno esincane sokukhulisa, inani lemijikelezo lingakhushulwa ngokufanelekile libe imijikelezo engama-40 -50.
Izinhlelo zokusebenza
Isebenza ekukhuliseni okuqondile kwezicubu zezitshalo kanye nokuhlolwa komphumela ophezulu wamasampula ezitshalo angaqukethe ama-polysaccharides nama-polyphenols.
Amanothi
Okokusetshenziswa kocwaningo kuphela.Ayisetshenziselwa izinqubo zokuxilonga.
1. Ukukhulisa izitshalo ezingahluziwe noma ukukhulisa ngokuqondile, kunconywa ukusebenzisa i-purified genomic DNA njengokulawula okuhle ngaphambi kokuqala ukuhlola ukuze kuqinisekiswe ukuthi isistimu, ama-primers kanye nokusebenza kulungile.
2. I-DNA polymerase ye-amplification eqondile esetshenziswe kule khithi inomsebenzi wokuhlola oqinile.Uma i-TA cloning idinga ukwenziwa, kunconywa ukuhlanza i-DNA ngaphambi kokwengeza i-adenine.
3. I-Primer Design Isiqondiso:
a.Kunconywa ukuthi isisekelo sokugcina ekupheleni kuka-3′ kufanele kube u-G noma u-C.
b.Ukungafani okulandelanayo kufanele kugwenywe kuzisekelo ezingu-8 zokugcina ekupheleni kuka-3′ kwesiqalo.c.Gwema izakhiwo ze-hairpin ekupheleni kuka-3′ kwe-primer.
d.Umehluko enanini le-Tm le-primer eya phambili kanye ne-primer ehlehlayo akufanele ibe ngaphezu kuka-1℃ futhi inani le-Tm kufanele lilungiswe libe ngu-60 ~ 72℃ (I-Primer Premier 5 iyanconywa ukubala inani le-Tm).
e.Ukulandelana okwengeziwe kwe-primer eyengeziwe okungafani nesifanekiso, akufanele kufakwe lapho kubalwa inani lokuqala le-Tm.
f.Kunconywa ukuthi okuqukethwe kwe-GC kwe-primer kube ngu-40% -60%.
g.Ukusabalalisa okuphelele kuka-A, G, C kanye no-T ku-primer kufanele kube ngokulingana ngangokunokwenzeka.Gwema ukusebenzisa izifunda ezinokuqukethwe okuphezulu kwe-GC noma kwe-AT.
h.Gwema ukuba khona kokulandelana okulandelanayo kwezisekelo ezingu-5 noma ngaphezulu phakathi kwe-primer noma phakathi kwama-primer amabili futhi ugweme ukuba khona kokulandelana okulandelanayo kwezisekelo ezingu-3 noma ngaphezulu ekupheleni kuka-3′ wama-primer amabili.
i.Sebenzisa umsebenzi we-NCBI BLAST ukuze uhlole ukucaciswa kwe-primer ukuvimbela ukukhulisa okungaqondile.
