Dyara yakananga PCR Kit
Katsi Nhamba: HCR2020A
Plant Direct PCR Kit inokodzera kukwidziridzwa kwakananga kwemashizha ezvirimwa, mhodzi, nezvimwewo, uye inogona kushandiswa pakakwirira-kuburikidza kuongororwa kwemasampuri echirimwa asina polysaccharides uye polyphenols.Iyo yakananga amplification DNA polymerase yakavakirwa pane yakatungamirwa shanduko ine kushivirira kwepamusoro kune PCR inhibitors muzvirimwa.Zvichakadaro, inochengetedza yakakwira amplification performance, iyo inokodzera kukwidziridzwa kwezvidimbu zveDNA mukati me5 kb.Iyo yakasarudzika yeLysis buffer A mukiti inogona kushandiswa lyse nyowani kana chando matishu ezvirimwa.Zviri nyore kushanda uye lysate inogona kushandiswa se template yekukudza pasina kucheneswa.Iyo sisitimu ine zvinodzivirira zvinogonesa crude samples kuti inyatso kukwidziridzwa mushure mekudzokororwa nechando uye kunyungudika.2 × Plant Direct Master Mix inongoda kuwedzera maprimers uye matemplate kuti aite amplification reaction, nekudaro kuderedza mashandiro epombi nekuvandudza mabudiro ekuona uye kuberekazve kwemhedzisiro.
Zvikamu
| Zvikamu | 50 RXNS | 200 RXNS |
| 2 × Plant Direct Master Mix | 1.25 ml | 4 × 1.25 ml |
| Dyara Yakananga Lysis Buffer A | 5 ml | 20 ml |
| Dyara Yakananga Lysis Buffer B* | 5 ml | 20 ml |
*Dyara Yakananga Lysis Buffer B isarudzo yeagent, iyo inoshandiswa kudzikamisa Plant Direct Lysis Buffer A yekurebesa nguva yekuchengetedza yemasampuli.Inogona kushandiswa maererano nemamiriro ezvinhu chaiwo.
Storage Conditions
2 × Plant Direct Master Mix, chengetedza pa -30 ~ -15 ℃ uye dzivisa kudzokorora kutonhora uye kunyunguduka;Dyara Yakananga Lysis Buffer, chitoro pa -30 ~ -15 ℃ kana 2 ~ 8 ℃.
Experiment Process
Muenzaniso Processing-Plant Leaf
Yakananga nzira:Inokurudzirwa kushandisa mashizha maduku.Shandisa gomba punch ine dhayamita yakatarwa ye0.5 - 3 mm kuti uwane diki uye yunifomu samplea, uye wobva wawedzera zvakananga sampu kuPCR system (50 μl system inokurudzirwa).Cherechedza, ita shuwa kuti sampuli iri muPCR mhinduro uye kwete pamadziro echubhu.Kana yakananga PCR inoshandiswa kukudza zvimedu zvenguva refu uye sampuli dzakaoma, kushandisa sampuli ine dhayamita diki (0.5 - 1 mm) se template inogona kubatsira kuwana mhedzisiro iri nani.
Kukuya lysis nzira:Inokurudzirwa kushandisa mashizha maduku.Tora chidimbu chidiki cheshizha (chinenge 1 - 3 mm muhupamhi), chiise mu20 μl Plant Direct Lysis Buffer Ab, woikuya nepaunogona napo (danho iri rinogona kuitwa nekusvina shizha nepipette 100 μl. kukuya sampuli).Kana mavhoriyamu mahombe ematishu emashizha akashandiswa (usapfuure 7 mm), wedzera huwandu hwe dilution buffer kusvika pa50 μl.Mushure mokunge mashizha ari pasi, mhinduro inofanira kuonekwa yakasvibirira.Mushure mechinguva chidiki centrifugation, wedzera 1 μl ye supernatant kune PCR system seyekuita templatec.
Thermal lysis nzira:Inokurudzirwa kushandisa mashizha maduku.Tora chidimbu chidiki cheshizha (chinenge 1 - 3 mm muhupamhi), chiise mu 20 μl Plant Direct Lysis Buffer A, uye inopisa pa 95 ° C kwe 5 - 10 min.Iyo nguva ye lysis inogona kuwedzerwa zvakakodzera kune mashizha akaoma lyse (kwete kupfuura 20 min).Kana mavhoriyamu akakura ematishu emashizha akashandiswa (usapfuure 7 mm), wedzera huwandu hwe dilution buffer kusvika pa50 μl.Mushure mekupisa, chengetedza zvishoma, uye wedzera 1 μl ye supernatant kune PCR system seyekuita templateb.
Muenzaniso Processing– Dyara Mbeu
Kukuya lysis nzira:Shandisa scalpel kucheka mhodzi dzine dhayamita 5 mm, woiisa ku100 μl yePlant Direct Lysis Buffer A, wokuya sample yacho nepipette tip kana mamwe maturusi.Vortex muchidimbu uye regai imire pakamuri tembiricha 3 - 5 min.Ita shuwa kuti sampuli yembeu yakanyura mudilution buffer.Mushure mechinguva chidiki centrifugation, wedzera 1 μl yemushura kuPCR system seyekuita template.
Thermal lysis nzira:Shandisa scalpel kucheka mhodzi dzine dhayamita 5 mm, woisa ku100 μl yePlant Direct Lysis Buffer A, uye kupisa pa95°C kwema5 - 10 min.Iyo nguva ye lysis inogona kuwedzerwa zvakakodzera kune mashizha akaoma lyse (kwete kupfuura 30 min).Mushure mekudziya, chengetedza zvishoma, uye wedzera 1 μl supernatant kune PCR system seyekuita templateb.
a.Zvigero kana mamwe maturusi anogonawo kushandiswa kucheka masampuli ehukuru hwakakodzera;kana chigero kana chigero chikashandiswazve, chinofanira kucheneswa ne 2% sodium hypochlorite mhinduro isati yashandiswa yega yega kudzivirira kusvibiswa kwepakati pakati pemasampuli.
b.Ita shuwa kuti Plant Direct Lysis Buffer yanyungudika zvizere isati yashandiswa.Kana iyo buffer iri viscous kana iine mvura inonaya, inogona kudziyisa pa 37 ℃ kuinyungudusa zvachose isati yashandiswa.
c.Vhoriyamu yetemplate mune rekuita system inogona kugadziridzwa zvakakodzera zvichienderana nemusiyano wehuwandu hwechirimwa zvinhu uye diluent yakawedzerwa.
Dyara Yakananga Lysis Buffer
Iyo Plant Direct Lysis Buffer A iri muchigadzirwa ichi yakagadziridzwa zvakanyanya kuburitsa genome yezvidyarwa zvakawanda uye inokodzera kuchengetwa kwenguva pfupi kwezvidyarwa zvisina kuchena pa4 ℃.Kana sampuli ichida kuchengetwa kwenguva yakareba (semuenzaniso, 1 - 2 mwedzi), inokurudzirwa kuendesa iyo supernatant kune itsva EP chubhu uye kuichengeta pa -20 ℃.Kuti uchengetedze masampuli zvakanyanya kugadzikana, wedzera yakaenzana vhoriyamu yePlant Direct Lysis Buffer B kune yakatamiswa mashura, sanganisa zvakanaka uye chengetedza pa -20 ℃.Iyo yakagadzika yekuchengetedza nguva inosiyana nezvirimwa samples uye nyika.
Reaction System
| ddH2O | Kusvika ku20.0µl | Kusvika ku50.0µl |
| 2 × Plant Direct Master Mixa | 10.0µl | 25.0µ |
| Primer 1 (10 µM) | 0.8µl | 2.0 µl |
| Primer 2 (10 µM)b | 0.8µl | 2.0 µl |
| Dyara mashizha / crude extract sample(Ona Sample Processing) | 0.5 - 3 mm leaf disc/x µl | 0.5 - 3 mm leaf disc/x µl |
a.Iyo ine Mg2+pachikamu chekupedzisira che 2 mM.
b.Inokurudzirwa kushandisa chiyero chekupedzisira che 0.4μM kune imwe neimwe primer.Kunyanya kushandisa maprimers kunotungamira kuwedzera kusingatarisike amplification.
c.Nhamba yemuenzaniso inoshandiswa inogona kugadziriswa maererano nemamiriro chaiwo.Huwandu hunoshandiswa mukuita kumwe kweiyo crude lysed sampu inogona kugadziriswa pakati pe2% - 20% yehuwandu hwehuwandu hwekuita.Kushandisa sampuli dzakawandisa kunogona kukonzera kutadza kwekusimudzira.
Reaction Program
| Matanho | Tembiricha | Nguva |
| Kutanga Denaturation | 98℃ | 5 min |
| Denaturation | 95℃ | 10 sec |
| Annealing | 58 ~ 72℃ | 15 sec |
| Extension | 72℃ | 30 sec |
| Final Extension | 72℃ | 5 min |
a.Initial Denaturation (98 ℃, 5 min) inosimudzira lysis yemiti yezvirimwa, kuburitsa genomic DNA inogona kushandiswa PCR amplification.Usapfupisa nguva kana kuderedza tembiricha.
b.Inokurudzirwa kuiisa yakaenzana neyekutanga Tm kukosha kana 2 ~ 4 ℃ yakakwirira kupfuura kukosha kweTm.Iyo yakananga amplification DNA polymerase inoshandiswa muchigadzirwa ichi yakasiyana neyakajairwa Taq DNA polymerase, uye ine zvakakosha zvinodiwa pakuita annealing tembiricha; kushandiswa kweyepamusoro tembiricha yekudziya kunogona kunyatso kudzikisa nonspecific amplification uye kugadzirisa amplification kunyatsoita.Kune yakaoma matemplate, iyo inobudirira amplification inogona kuwanikwa nekugadzirisa annealing tembiricha uye kuwedzera nguva yekuwedzera.
c.Kana kureba kwechigadzirwa chekusimudzira chiri ≤1 kb, nguva yekuwedzera yakaiswa pa 30 sec/kb;kana kureba kwechigadzirwa chekusimudzira chiri> 1 kb, nguva yekuwedzera inoiswa pa60 sec/kb.
d.Kune dzakaomarara sampuli kana masampula ane yakaderera amplification goho, huwandu hwematenderedzwa hunogona kukwidziridzwa zvakakodzera kusvika ku40 -50 cycles.
Applications
Inoshanda kune yakananga kukwidziridzwa kwemiti yezvirimwa uye yakakwira-kuburikidza kuongororwa kwemasampuri echirimwa asina mapolysaccharides uye polyphenols.
Notes
Yekushandisa tsvakurudzo chete.Kwete kushandiswa mukuongorora maitiro.
1. Kuti crude plant amplification kana yakananga amplification, zvinokurudzirwa kushandisa purified genomic DNA sechinhu chakanaka chekutonga usati watanga kuedza kuti uone kuti system, primers uye kushanda kwakarurama.
2. Iyo yakananga amplification DNA polymerase inoshandiswa mukiti iyi ine simba rekuverenga basa.Kana TA cloning ichida kuitwa, zvinokurudzirwa kuchenesa DNA usati wawedzera adenine.
3. Primer Design Guidance:
a.Zvinokurudzirwa kuti chigadziko chekupedzisira pa3 ′ kupera kweprimer chinofanirwa kunge chiri G kana C.
b.Kusawirirana kunotevedzana kunofanirwa kudzivirirwa mumabhesi masere ekupedzisira pa3 ′ kupera kweprimer.c.Dzivisa zvimiro zvehairpin pa3 ′ kumagumo ekutanga.
d.Misiyano mumutengo weTm weiyo yekutanga primer uye reverse primer haifanirwe kudarika 1 ℃ uye kukosha kweTm kunofanirwa kugadziridzwa kusvika 60 ~ 72 ℃ (Primer Premier 5 inokurudzirwa kuverenga kukosha kweTm).
e.Yekuwedzera yakawedzera kutevedzana kwekutanga iyo isingaenderane netemplate, haifanirwe kuverengerwa pakuverenga iyo yekutanga Tm kukosha.
f.Inokurudzirwa kuti iyo GC yemukati yeprimer ive 40% -60%.
g.Kugoverwa kwese kweA, G, C uye T mukutanga kunofanirwa kuve kwakatodzana sezvinobvira.Dzivisa kushandisa matunhu ane yakakwira GC kana AT zvirimo.
h.Dzivisa kuvapo kwekutevedzana kwekutevedzana kwe5 kana kupfuura mabhesi angave mukati mekutanga kana pakati pemaprimers maviri uye dzivirira kuvapo kwekutevedzana kwekutevedzana kwemabhesi matatu kana anopfuura pa3′ kupera kwemaprimers maviri.
i.Shandisa iyo NCBI BLAST basa kuti utarise iwo chaiwo eprimer kudzivirira nonspecific amplification.
