Tera PCR Kit

Injangwe No: HCR2020A

Igiterwa Direct PCR Kit gikwiranye no kongera amababi y ibihingwa, imbuto, nibindi, kandi birashobora gukoreshwa mugupima cyane kwerekanwa ryibihingwa bitarimo polysaccharide na polifenol.Kwiyongera kwa ADN polymerase gushingiye ku bwihindurize bwerekanwe bifite kwihanganira cyane PCR ibuza ibimera.Hagati aho, ikomeza imikorere yo hejuru cyane, ikwiranye no kongera ibice bya ADN muri kb 5.Buffer idasanzwe ya Lysis A mubikoresho irashobora gukoreshwa mugutobora ibimera bishya cyangwa bikonje.Biroroshye gukora kandi lysate irashobora gukoreshwa nkicyitegererezo cyo kwongera imbaraga nta kweza.Sisitemu ikubiyemo ibintu birinda ibintu bituma ingero zidasanzwe zongerwa neza nyuma yo gukonjesha no gukonja.2 × Gutera Direct Master Master ivanze ikeneye gusa kongeramo primers na templates kugirango ikore amplification reaction, bityo bigabanye ibikorwa byo kuvoma no kunoza uburyo bwo gutahura no kubyara ibisubizo.

Ibigize

* Gutera Lysis Buffer B ni B reagent itabishaka, ikoreshwa muguhindura ibimera bya Lysis Buffer A kugirango byongere igihe cyo kubika ingero.Irashobora gukoreshwa ukurikije uko ibintu bimeze.

Ububiko

2 × Tera Master Master Mix, ubike kuri -30 ~ -15 ℃ kandi wirinde gukonjesha inshuro nyinshi;Tera Lysis Buffer, ubike kuri -30 ~ -15 ℃ cyangwa 2 ~ 8 ℃.

Inzira y'Ubushakashatsi

Icyitegererezo-Gutera amababi

Uburyo butaziguye:Birasabwa gukoresha amababi akiri mato.Koresha umwobo ufite umurambararo wa 0.5 - 3 mm kugirango ubone samplea ntoya kandi imwe, hanyuma wongereho icyitegererezo muri sisitemu ya PCR (birasabwa 50 μl sisitemu).Icyitonderwa, menya neza ko icyitegererezo kiri mubisubizo bya PCR kandi bitarwanya urukuta.Niba PCR itaziguye ikoreshwa muguhuza ibice birebire hamwe nurugero rugoye, ukoresheje icyitegererezo gifite diameter ntoya (0.5 - 1 mm) nkicyitegererezo gishobora gufasha kubona ibisubizo byiza.

Gusya lysis uburyo:Birasabwa gukoresha amababi akiri mato.Fata agace gato k'ibabi (hafi 1 - 3 mm z'umurambararo), ubishyire muri 20 μl Plant Direct Lysis Buffer Ab, hanyuma ubisya bishoboka (iyi ntambwe irashobora gukorwa no gukanda ikibabi ukoresheje 100 μl pipette gushisha icyitegererezo).Niba ingano nini yimyenda yamababi yakoreshejwe (nturenze mm 7), ongera ingano ya buffer ya dilution kugeza kuri 50 μl.Amababi amaze kuba hasi, igisubizo kigomba kugaragara icyatsi.Nyuma ya centrifugation ngufi, ongeramo 1 μl yindengakamere kuri sisitemu ya PCR nkicyitegererezo.

Uburyo bwa lysis yubushyuhe:Birasabwa gukoresha amababi akiri mato.Fata agace gato k'ibabi (hafi mm 1 - 3 z'umurambararo), ubishyire muri 20 μl Igiterwa cya Lysis Buffer A, hanyuma ubishyuhe kuri 95 ° C muminota 5 - 10.Igihe cya lysis kirashobora kwongerwa muburyo bukwiye kubibabi bigoye kurigata (bitarenze min 20).Niba ingano nini yimyenda yamababi yakoreshejwe (nturenze mm 7), ongera ingano ya buffer ya dilution kugeza kuri 50 μl.Nyuma yo gushyushya, centrifuge muri make, hanyuma ongeramo 1 μl yindengakamere muri sisitemu ya PCR nkicyitegererezo.

Icyitegererezo- Imbuto

Gusya lysis uburyo:Koresha scalpel kugirango ukate imbuto zifite umurambararo wa mm 5, ubyongereho kuri 100 μl za Plant Direct Lysis Buffer A, hanyuma usya icyitegererezo ukoresheje umuyoboro wa pipette cyangwa ibindi bikoresho.Vortex muri make hanyuma ureke guhagarara mubushyuhe bwicyumba cya 3 - 5 min.Menya neza ko icyitegererezo cyimbuto cyarengewe muri buffer ya dilution.Nyuma ya centrifugation ngufi, ongeramo 1 μl yindengakamere kuri sisitemu ya PCR nkicyitegererezo.

Uburyo bwa lysis yubushyuhe:Koresha scalpel kugirango ukate imbuto zifite umurambararo wa mm 5, ongeraho kuri 100 μl za Plant Direct Lysis Buffer A, hanyuma ushushe kuri 95 ° C kuminota 5 - 10.Igihe cya lysis kirashobora kwongerwa muburyo bukwiye kubibabi bigoye kurigata (bitarenze min 30).Nyuma yo gushyushya, centrifuge muri make, hanyuma ongeramo 1 μl ndengakamere muri sisitemu ya PCR nkicyitegererezo.

a.Imikasi cyangwa ibindi bikoresho nabyo birashobora gukoreshwa mugukata ingero zingana;niba igikoni cyangwa imikasi byongeye gukoreshwa, bigomba guhanagurwa hamwe na 2% sodium hypochlorite yumuti mbere yo gukoreshwa kugirango birinde kwanduzanya hagati yintangarugero.

b.Menya neza ko Igiti cya Lysis Buffer yashonga mbere yo kuyikoresha.Niba buffer ifite ibara ryinshi cyangwa ifite imvura, irashobora gushyuha kuri 37 ℃ kuyishonga burundu mbere yo kuyikoresha.

c.Ingano yicyitegererezo muri sisitemu ya reaction irashobora guhindurwa uko bikwiye ukurikije itandukaniro ryubunini bwibikoresho byibimera hamwe na diluent yongeyeho.

Tera Lysis Buffer

Igihingwa cya Lysis Buffer A gikubiye muri iki gicuruzwa cyarahinduwe neza kugirango kirekure genome yingirangingo nyinshi z’ibimera kandi kibereye kubika igihe gito ibihingwa bitavanze kuri 4 ℃.Niba icyitegererezo gikeneye kubikwa mugihe kirekire (urugero, amezi 1 - 2), birasabwa kohereza supernatant kumuyoboro mushya wa EP hanyuma ukabika kuri -20 ℃.Kubika ibyitegererezo neza, ongeramo ingano ingana na Plant Direct Lysis Buffer B kuri supernatant yimuwe, vanga neza hanyuma ubike kuri -20 ℃.Igihe cyo kubika gihamye kiratandukanye nicyitegererezo cyibimera na leta.

Sisitemu yo Kwitabira

a.Harimo Mg²⁺kuri concentration ya nyuma ya 2 mM.

b.Birasabwa gukoresha intumbero yanyuma ya 0.4μM kuri buri primer.Gukoresha cyane primers bizaganisha ku kwiyongera kwa amplification idafite akamaro.

c.Ingano yicyitegererezo yakoreshejwe irashobora guhinduka ukurikije uko ibintu bimeze.Amafaranga yakoreshejwe muburyo bumwe bwa sample ya lysed sample irashobora guhinduka hagati ya 2% - 20% yubunini bwa reaction.Gukoresha ingero nyinshi birashobora gutera kunanirwa kwa amplification.

Gahunda yo Kwitabira

a.Gutandukana kwambere (98 ℃, 5 min) biteza lysis yumubiri wibimera, kurekura ADN genomic ishobora gukoreshwa muguhindura PCR.Ntugabanye igihe cyangwa ngo ugabanye ubushyuhe.

b.Birasabwa gushiraho bingana na primer Tm agaciro cyangwa 2 ~ 4 ℃ hejuru ya Tm agaciro.Kwiyongera kwa ADN polymerase ikoreshwa muri iki gicuruzwa bitandukanye na polymerase isanzwe ya Taq ADN, kandi ifite ibisabwa byihariye kugirango ubushyuhe bwa annealing ； gukoresha ubushyuhe bwo hejuru bwa annealing burashobora kugabanya neza kwongerera imbaraga no kunoza imikorere.Kubishusho byoroshye, amplification ikora neza irashobora kugerwaho muguhindura ubushyuhe bwa annealing no kongera igihe cyo kwagura.

c.Niba uburebure bwibicuruzwa byongerewe ari ≤1 kb, igihe cyo kwagura gishyirwaho 30 sec / kb;niba uburebure bwibicuruzwa byongerewe ari> 1 kb, igihe cyo kwagura gishyirwa kuri 60 sec / kb.

d.Kuburugero rwicyitegererezo cyangwa ingero zifite umusaruro muke wa amplification, umubare wizunguruka urashobora kwiyongera muburyo bukwiye kugeza kuri 40 -50.

Porogaramu

Irakoreshwa muburyo bwo kongera imbaraga mu buryo butaziguye ingirabuzima fatizo no gusuzuma-urugero rwinshi rw’ibitegererezo by'ibimera bitarimo polysaccharide na polifenol.

Inyandiko

Kubushakashatsi ukoreshe gusa.Ntabwo ari ugukoresha muburyo bwo gusuzuma.

1. Kubijyanye no kongera ibimera byangiritse cyangwa kwongerwaho imbaraga, birasabwa gukoresha ADN ya genomic yasukuwe nkigenzura ryiza mbere yo gutangira igerageza kugirango sisitemu, primers nibikorwa bikosore.

2. Amplifisione itaziguye ya ADN polymerase ikoreshwa muriki gikoresho ifite ibikorwa bikomeye byo gusuzuma.Niba hakenewe gukorwa cloni ya TA, birasabwa kweza ADN mbere yo kongeramo adenine.

3. Amabwiriza agenga ibishushanyo mbonera ：

a.Birasabwa ko shingiro ryanyuma kuri 3 ′ impera ya primer igomba kuba G cyangwa C.

b.Kudahuza gukurikiranye bigomba kwirindwa mubice 8 byanyuma kuri 3 ′ iherezo rya primer.c.Irinde imiterere yimisatsi kuri 3 ′ iherezo rya primer.

d.Itandukaniro muri Tm agaciro kambere primer na revers primer ntigomba kurenza 1 ℃ kandi agaciro ka Tm kagomba guhinduka kuri 60 ~ 72 ℃ (Primer Premier 5 irasabwa kubara agaciro ka Tm).

e.Ibindi byongeweho primer bikurikiranye bidahuye nicyitegererezo, ntibigomba kubamo mugihe ubara primer Tm agaciro.

f.Birasabwa ko GC yibigize primer kuba 40% -60%.

g.Gukwirakwiza muri rusange A, G, C na T muri primer bigomba kuba nubwo bishoboka.Irinde gukoresha uturere dufite GC nyinshi cyangwa AT.

h.Irinde kuba hari urutonde rwuzuzanya rwibintu 5 cyangwa byinshi haba muri primer cyangwa hagati ya primers ebyiri kandi wirinde ko habaho urutonde rwuzuzanya rwibintu 3 cyangwa byinshi kuri 3 ′ iherezo rya primers ebyiri.

i.Koresha imikorere ya NCBI BLAST kugirango ugenzure umwihariko wa primer kugirango wirinde amplification idafite akamaro.