Nka shyashya muri laboratoire, ntabwo ari akazi keza kugenzura ibihingwa byiza bivuye kumurima wibimera bifite igipimo gito cyo guhinduka.Ubwa mbere, ADN igomba gukurwa mububare bwinshi bw'icyitegererezo umwe umwe, hanyuma genes zo mumahanga zikamenyekana na PCR.Nyamara, ibisubizo akenshi usanga ari ubusa hamwe na bande hamwe nibintu bike rimwe na rimwe, ariko ntibishoboka kumenya niba hari wabuze gutahura cyangwa gutahura ibinyoma..Ntibishoboye cyane guhangana nuburyo bwo kugerageza nibisubizo?Ntugire ikibazo, muvandimwe akwigisha uburyo bwo kwerekana ibimera byiza bya transgenji byoroshye kandi neza.

Intambwe ya 1

Igishushanyo mbonera

Menya gene ya endogenous na gene ya exogenous igomba kumenyekana ukurikije icyitegererezo kizageragezwa, hanyuma uhitemo uhagarariye 100-500bp ikurikiranye muri gene kugirango ibe primer.Intangiriro nziza irashobora kwemeza neza ibisubizo byo gutahura no kugabanya igihe cyo gutahura (reba umugereka wibisanzwe bikoreshwa mugushakisha).

Icyitonderwa: Ibishushanyo mbonera byateguwe bigomba guhindura uburyo bwo kubyitwaramo no kugenzura niba imipaka igaragara neza, mbere na mbere.

Intambwe ya 2

Shushanya protocole yubushakashatsi

Igenzura ryiza: Koresha ADN isukuye irimo igice cyerekanwe nkicyitegererezo kugirango umenye niba sisitemu ya PCR nuburyo bisanzwe.

Igenzura ribi / ryuzuye: Koresha icyitegererezo cya ADN cyangwa ddH2O itarimo igice cyerekanwe nkicyitegererezo kugirango umenye niba hari isoko yanduye muri sisitemu ya PCR.

Igenzura ryimbere mu gihugu: koresha primer / probe ihuza gene ya endogenous ya sample kugirango igerageze gusuzuma niba inyandikorugero ishobora gutahurwa na PCR.

Icyitonderwa:

Igenzura ryiza, ribi / ryuzuye nubugenzuzi bwimbere bigomba gushyirwaho kuri buri kizamini kugirango harebwe agaciro k ibisubizo byubushakashatsi.

Gutegura ubushakashatsi

Mbere yo gukoresha, reba niba igisubizo kivanze.Niba imvura ibonetse, igomba gushonga no kuvangwa ukurikije amabwiriza mbere yo kuyakoresha.2 mix PCR ivanze igomba guhindurwa no kuvangwa inshuro nyinshi na micropipette mbere yo kuyikoresha kugirango wirinde gukwirakwiza ion kutaringaniye.

Icyitonderwa:

Kuramo imfashanyigisho uyisome witonze, hanyuma witegure mbere yikigeragezo ukurikije ibisabwa nigitabo.

Intambwe ya 4

Tegura sisitemu ya reaction ya PCR

Ukurikije protocole yubushakashatsi, vanga primers, H2O, na 2 × PCR bivanga neza, centrifuge hanyuma ubigabanye kuri buri tube reaction.

Icyitonderwa:

Kubizamini binini cyangwa birebire, birasabwa gukoresha sisitemu ya PCR irimo enzyme ya UNG, ishobora kwirinda neza kwanduza aerosol iterwa nibicuruzwa bya PCR.

Intambwe ya 5

Ongeraho inyandikorugero

Ukoresheje tekinoroji ya PCR itaziguye, ntihakenewe uburyo bwo kweza aside nucleic iruhije, icyitegererezo cyicyitegererezo gishobora gutegurwa muminota 10, kandi sisitemu ihuye na PCR irashobora kongerwamo.

Icyitonderwa:

Uburyo bwa clavage bufite ingaruka nziza zo gutahura, kandi ibicuruzwa byabonetse birashobora gukoreshwa mubitekerezo byinshi.

5.1: Kwaguka kwamababi

Ukurikije ubunini bwifoto iri mu gitabo, gabanya ingirangingo yamababi hamwe na diameter ya 2-3mm hanyuma ubishyire muri sisitemu ya reaction ya PCR.

Icyitonderwa: Menya neza ko ibice byamababi byinjijwe rwose mubisubizo bya PCR, kandi ntukongereho ibibabi bikabije.

5.2: Uburyo bwo gutandukanya amababi

Kata ibibabi byamababi hamwe na diameter ya 5-7mm hanyuma ubishyire mumiyoboro ya centrifuge.Niba uhisemo amababi akuze, nyamuneka wirinde gukoresha ingirangingo z'imitsi nyamukuru y'ibabi.Pipette 50ul Buffer P1 lysate mumiyoboro ya centrifuge kugirango urebe ko lysate ishobora kwibiza rwose ibibabi byamababi, ikabishyira mumagare yumuriro cyangwa ubwogero bwicyuma, hanyuma lyse kuri 95 ° C muminota 5-10.

Ongeramo 50ul Buffer P2 igisubizo cyo kutabogama hanyuma uvange neza.Lysate yavuyemo irashobora gukoreshwa nkicyitegererezo kandi ikongerwa kuri sisitemu ya reaction ya PCR.

Icyitonderwa: Ingano yicyitegererezo iri hagati ya 5-10% ya sisitemu ya PCR, kandi ntigomba kurenga 20% (urugero, muri sisitemu ya 20μl PCR, ongeramo 1-2μl yumuti wa lysis, ntabwo urenze 4μl).

Intambwe ya 6

PCR reaction

Nyuma ya centrifuging ya reaction ya PCR, ishyirwa mubikoresho bya PCR kugirango amplification.

Icyitonderwa:

Igisubizo gikoresha inyandikorugero idasukuwe kugirango yongerwe imbaraga, bityo umubare wikizunguruka cyikubye inshuro 5-10 kurenza iyo ukoresheje ADN isukuye.

Intambwe 7

Kugaragaza amashanyarazi no gusesengura ibisubizo

M: 100bp Urwego rwa ADN

1 \ 4: Uburyo bwa ADN bwejejwe

2 \ 5: Uburyo bwa PCR butaziguye

3 \ 6: Kugenzura neza

QC:

Ibisubizo by'ibizamini byubugenzuzi butandukanye bwashyizweho mubigeragezo bigomba kuba byujuje ibi bikurikira.Bitabaye ibyo, icyateye ikibazo kigomba gusesengurwa, kandi ikizamini kigomba kongera gukorwa nyuma yikibazo.

Imbonerahamwe 1. Ibisubizo bisanzwe byikizamini cyamatsinda atandukanye yo kugenzura

* Iyo plasmid ikoreshwa nkigenzura ryiza, ibisubizo bya endogenous gene ibisubizo birashobora kuba bibi

Urubanza:

Igisubizo.

B. Ibisubizo by'ibizamini bya gene ya endogenous y'icyitegererezo ni byiza, kandi ibisubizo by'ibizamini bya gene exogenous ni bibi, byerekana ko ADN ikwiranye na PCR isanzwe ikurwa mu cyitegererezo, kandi hashobora kugaragara ko gene ya XXX itabonetse muri sample.

C. Ibisubizo by'ibizamini bya gene ya endogenous y'icyitegererezo ni byiza, kandi ibisubizo by'ibizamini bya gene exogenous ni byiza, byerekana ko ADN ikwiranye na PCR isanzwe yakuwe mu cyitegererezo, kandi ADN y'icyitegererezo irimo gene ya XXX.Ubushakashatsi bwo kwemeza burashobora gukorwa.

Intambwe ya 8

Igishushanyo mbonera

Nyuma yubushakashatsi, koresha 2% sodium hypochlorite yumuti na 70% yumuti wa Ethanol kugirango uhanagure ahakorerwa ubushakashatsi kugirango wirinde kwanduza ibidukikije。