Tyala PCR Kit ngqo
Inombolo yekati: HCR2020A
I-Plant Direct PCR Kit ifanelekile ukukhulisa ngokuthe ngqo amagqabi ezityalo, imbewu, njl., kwaye ingasetyenziselwa ukuhlolwa okuphezulu kweesampuli zezityalo ezingenayo i-polysaccharides kunye ne-polyphenols.I-DNA polymerase yokukhulisa ngokuthe ngqo esekelwe kwi-evolution ekhokelwayo inokunyamezela okuphezulu kwi-PCR inhibitors kwizityalo.Okwangoku, igcina ukusebenza kokukhulisa okuphezulu, okufanelekile ukukhulisa amaqhekeza e-DNA ngaphakathi kwe-5 kb.I-Lysis buffer ekhethekileyo ye-A kwikhithi ingasetyenziselwa ukukhupha izicubu zezityalo ezitsha okanye ezikhenkcezisiweyo.Kulula ukusebenza kwaye i-lysate ingasetyenziselwa njenge template yokukhulisa ngaphandle kokuhlanjululwa.Inkqubo iqulethe ii-agent ezikhuselayo ezenza ukuba iisampulu ezikrwada zandiswe ngokufanelekileyo emva komkhenkce ngokuphindaphindiweyo kunye nokunyibilika.I-2 × I-Plant Direct Master Mix idinga kuphela ukongeza ii-primers kunye neetemplates ukwenza i-amplification reaction, ngaloo ndlela inciphisa imisebenzi yepayipi kunye nokuphucula ukufumanisa ukukhutshwa kunye nokuveliswa kweziphumo.
Amacandelo
| Amacandelo | 50 RXNS | 200 RXNS |
| I-2 × I-Plant Direct Master Mix | 1.25 ml | 4×1.25 ml |
| Tyala ngokuthe ngqo uLysis Buffer A | 5 ml | 20 ml |
| Tyala ngokuthe ngqo iLysis Buffer B* | 5 ml | 20 ml |
*Plant Direct Lysis Buffer B sisixhobo sokuphinda sisebenze, esisetyenziselwa ukwenza iplanti Direct Lysis Buffer A yokwandisa ixesha lokugcina iisampulu.Ingasetyenziswa ngokweyona meko.
Iimeko zokuGcina
2 × Plant Direct Master Mix, gcina kwi -30 ~ -15 ℃ kwaye ugweme ukukhenkceza ngokuphindaphindiweyo kunye nokunyibilika;Plant Direct Lysis Buffer, gcina -30 ~ -15℃ okanye 2 ~ 8℃.
Inkqubo yoVavanyo
Isample Processing-Igqabi lokuTyalwa
Indlela ethe ngqo:Kunconywa ukusebenzisa amaqabunga amancinci.Sebenzisa i-punch yomngxuma kunye nobubanzi obuqingqiweyo be-0.5 - 3 mm ukuze ufumane isampuli encinci kunye neyunifomu, uze udibanise ngokuthe ngqo isampuli kwinkqubo ye-PCR (i-50 μl inkqubo iyanconywa).Qaphela, qiniseka ukuba isampuli kwisisombululo se-PCR kwaye kungekhona ngokuchasene nodonga lombhobho.Ukuba i-PCR ethe ngqo isetyenziselwa ukukhulisa amaqhekeza amade kunye neesampuli eziyinkimbinkimbi, ukusebenzisa isampuli kunye nobubanzi obuncinci (0.5 - 1 mm) njenge template kunokunceda ukufumana iziphumo ezingcono.
Indlela yokugaya i-lysis:Kunconywa ukusebenzisa amaqabunga amancinci.Thatha iqhekezana legqabi (malunga ne-1 – 3 mm ububanzi), libeke kwi-20 μl Plant Direct Lysis Buffer Ab, kwaye ulisile kangangoko (eli nyathelo linokwenziwa ngokucudisa igqabi ngencam ye-100 μl yepipette. ukucuba isampuli) .Ukuba imithamo emikhulu yethishu yamagqabi isetyenzisiwe (ungadluli kwi-7 mm), yongeza umthamo we-dilution buffer ukuya kuma-50 μl.Emva kokuba amaqabunga aphantsi, isisombululo kufuneka sibonakale siluhlaza.Emva kwe-centrifugation emfutshane, yongeza i-1 μl ye-supernatant kwinkqubo ye-PCR njenge-reaction templatec.
Indlela ye-Thermal lysis:Kunconywa ukusebenzisa amaqabunga amancinci.Thatha iqhekeza elincinci legqabi (malunga ne-1 - 3 mm ububanzi), libeke kwi-20 μl Isityalo esiNgqongileyo seLysis Buffer A, kwaye siyifudumeze kwi-95 ° C nge-5 - 10 min.Ixesha le-lysis linokwandiswa ngokufanelekileyo kumagqabi anzima ukukhupha (akukho ngaphezu kwe-20 min).Ukuba imithamo emikhulu yethishu yamagqabi isetyenzisiwe (ungadluli kwi-7 mm), yongeza umthamo we-dilution buffer ukuya kuma-50 μl.Emva kokufudumeza, faka i-centrifuge ngokufutshane, kwaye wongeze i-1 μl ye-supernatant kwinkqubo ye-PCR njenge templateb yokusabela.
Isample Processing–Tyala iMbewu
Indlela yokugaya i-lysis:Sebenzisa i-scalpel ukusika imbewu enobubanzi obuyi-5 mm, wongeze kwi-100 μl ye-Plant Direct Lysis Buffer A, kwaye usile isampuli ngencam ye-pipette okanye ezinye izixhobo.I-Vortex ngokufutshane kwaye uvumele ukuma kwindawo yokushisa kwe-3 - 5 min.Qinisekisa ukuba isampulu yembewu intywiliselwe kwidilution buffer.Emva kwe-centrifugation emfutshane, yongeza i-1 μl ye-supernatant kwinkqubo ye-PCR njengetemplate yokusabela.
Indlela ye-Thermal lysis:Sebenzisa i-scalpel ukusika imbewu enobubanzi obuyi-5 mm, wongeze kwi-100 μl ye-Plant Direct Lysis Buffer A, kunye nobushushu obungama-95 ° C kangange-5 - 10 min.Ixesha le-lysis linokwandiswa ngokufanelekileyo kumagqabi anzima ukukhupha (akukho ngaphezu kwe-30 min).Emva kokufudumeza, faka i-centrifuge ngokufutshane, kwaye wongeze i-1 μl ephezulu kwi-PCR inkqubo njenge-templateb yokusabela.
a.Izikere okanye ezinye izixhobo zingasetyenziselwa ukusika iisampuli zobukhulu obufanelekileyo;ukuba i-punch okanye i-scissors iphinda isetyenziswe, kufuneka ihlambuluke nge-2% yesisombululo se-sodium hypochlorite ngaphambi kokusetyenziswa ngalunye ukukhusela ukungcola phakathi kweesampuli.
b.Qinisekisa ukuba i-Plant Direct Lysis Buffer inyibilike ngokupheleleyo phambi kokusetyenziswa.Ukuba isithinteli si-viscous okanye sinemvula, sinokufudunyezwa kwi-37℃ ukuyinyibilikisa ngokupheleleyo phambi kokusetyenziswa.
c.Umthamo wethempleyithi kwinkqubo yokusabela unokulungelelaniswa ngokufanelekileyo ngokomahluko kumthamo wezinto zezityalo kunye ne-diluent eyongeziweyo.
Tyala Ngqo Lysis Buffer
I-Plant Direct Lysis Buffer A equlethwe kule mveliso ilungiselelwe ngokungqongqo ukukhulula i-genome yezicubu ezininzi zezityalo kwaye ifanele ukugcinwa kwexesha elifutshane lezityalo ezikrwada kwi-4℃.Ukuba isampuli idinga ukugcinwa ixesha elide (umzekelo, 1 - 2 iinyanga), kucetyiswa ukuba udlulisele i-supernatant kwi-tube entsha ye-EP kwaye uyigcine kwi -20 ℃.Ukugcina iisampulu ngokuzinzileyo, yongeza umthamo olinganayo weSityalo esiNgqo kwiLysis Buffer B kwi-supernatant egqithiselweyo, xuba kakuhle kwaye ugcine ku -20℃.Ixesha lokugcina elizinzileyo lihluka kunye neesampuli zezityalo kunye namazwe.
Indlela yokusabela
| ddH2O | Ukuya kwi-20.0µl | Ukuya kwi-50.0µl |
| I-2 × I-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 |
| Isityalo segqabi / isampulu ekrwada yesicatshulwa(Jonga iSample Processing) | 0.5 – 3 mm leaf disc/x µl | 0.5 – 3 mm leaf disc/x µl |
a.Iqulethe uMg2+kugxininiso lokugqibela lwe-2 mM.
b.Kunconywa ukusebenzisa i-concentration yokugqibela ye-0.4μM kwi-primer nganye.Ukusetyenziswa kakhulu kweeprimers kuya kukhokelela ekwandisweni kokwandisa okungacaciswanga.
c.Isixa sesampuli esisetyenzisiweyo sinokuhlengahlengiswa ngokweyona meko.Isixa esisetyenziswe kwimpendulo enye yesampuli ekrwada ye-lysed inokulungelelaniswa phakathi kwe-2% - 20% yomthamo opheleleyo wokusabela.Ukusebenzisa iisampulu ezininzi kakhulu kunokubangela ukusilela kwe-amplification.
Inkqubo yokusabela
| Amanyathelo | Ubushushu | Ixesha |
| I-Denaturation yokuqala | 98℃ | 5 imiz |
| I-Denaturation | 95℃ | 10 imizuzwana |
| Ukuhlaziya | 58 ~ 72℃ | 15 imizuzwana |
| Ulwandiso | 72℃ | 30 imizuzwana |
| Ukwandiswa kokugqibela | 72℃ | 5 imiz |
a.I-Denaturation yokuqala (i-98℃, i-5 min) ikhuthaza i-lysis yezicubu zezityalo, ikhulula i-DNA ye-genomic engasetyenziselwa ukukhulisa i-PCR.Musa ukunciphisa ixesha okanye unciphise ubushushu.
b.Kucetyiswa ukuba uyibeke ilingane nexabiso le-primer Tm okanye i-2 ~ 4℃ ngaphezulu kwexabiso le-Tm.I-polymerase ye-DNA yokukhulisa ngokuthe ngqo esetyenziswe kule mveliso yahlukile kwi-polymerase ye-Taq DNA eqhelekileyo, kwaye ineemfuno ezikhethekileyo zobushushu bokusabela kwe-annealing;Kwiitemplates ezinzima, ukukhulisa okusebenzayo kunokufezekiswa ngokulungelelanisa ubushushu be-annealing kunye nokwandisa ixesha lokwandisa.
c.Ukuba ubude bemveliso yokukhulisa ngu-≤1 kb, ixesha elongezelelweyo libekwe kwi-30 sec / kb;ukuba ubude bemveliso yokukhulisa i> 1 kb, ixesha elongezelelweyo limiselwe kwi-60 sec/kb.
d.Kwiisampuli eziyinkimbinkimbi okanye iisampulu ezinemveliso ephantsi yokukhulisa, inani lemijikelezo inokunyuswa ngokufanelekileyo ukuya kuma-40 -50 imijikelezo.
Usetyenziso
Isebenza ngokukhuliswa ngokuthe ngqo kwezicubu zezityalo kunye nokuhlolwa okuphezulu kweesampuli zezityalo ezingenayo i-polysaccharides kunye ne-polyphenols.
Amanqaku
Isetyenziselwa uphando kuphela.Ayisetyenziswa kwiinkqubo zokuxilonga.
1. Ukukhulisa izityalo ezikrwada okanye ukukhulisa ngokuthe ngqo, kucetyiswa ukuba kusetyenziswe i-genomic DNA ehlanjululweyo njengolawulo olulungileyo ngaphambi kokuba uqalise uvavanyo lokuqinisekisa ukuba inkqubo, iiprimers kunye nokusebenza zichanekile.
2. I-DNA polymerase yokukhulisa ngokuthe ngqo esetyenziswe kule khithi inomsebenzi owomeleleyo wokuvavanya ubungqina.Ukuba i-TA cloning idinga ukwenziwa, kucetyiswa ukuba uhlambulule i-DNA ngaphambi kokuba ungeze i-adenine.
3. IsiKhokelo soYilo lokuqala:
a.Kunconywa ukuba isiseko sokugqibela kwi-3′ ekupheleni kwe-primer kufuneka ibe yi-G okanye i-C.
b.Ukungahambi kakuhle okulandelelanayo kufuneka kugwenywe kwiziseko ezi-8 zokugqibela kwi-3′ ekupheleni kwe-primer.c.Gwema izakhiwo ze-hairpin ekupheleni kwe-3 ye-primer.
d.Umahluko kwixabiso le-Tm le-primer yangaphambili kunye ne-primer ebuyela umva kufuneka ingabi ngaphezu kwe-1℃ kwaye ixabiso le-Tm kufuneka lihlengahlengiswe ukuya ku-60 ~ 72℃ (I-Primer Premier 5 iyacetyiswa ukubala ixabiso le-Tm).
e.Ulandelelwano lwe-primer olongezelelweyo olongezelelweyo olungahambelani nethempleyithi, akufanele lufakwe xa kubalwa ixabiso le-primer Tm.
f.Kucetyiswa ukuba umxholo weGC weprimer ube ngama-40% -60%.
g.Ukuhanjiswa ngokubanzi kwe-A, G, C kunye no-T kwi-primer kufuneka kube ngokulinganayo.Kuphephe ukusebenzisa imimandla ene-GC ephezulu okanye imixholo ye-AT.
h.Gwema ubukho bolandelelwano oluhambelanayo lwe-5 okanye iziseko ezingaphezulu nokuba ngaphakathi kwe-primer okanye phakathi kwee-primers ezimbini kwaye ugweme ubukho bokulandelelana okuhambelanayo kwe-3 okanye iziseko ezingaphezulu kwi-3′ ekupheleni kwee-primers ezimbini.
i.Sebenzisa umsebenzi we-NCBI BLAST ukujonga ukuchana kwe-primer ukuthintela ukukhulisa okungacaciswanga.
