Incamake
Kumenyekanisha byihuse ibimera bya transgenji
Inyandiko / Tong Yucheng
Igeragezwa / Han Ying
Muhinduzi / Wen Youjun
Amagambo / 1600 +
Igitekerezo cyo gusoma / iminota 8-10
Kumenyekanisha byihuse ibimera bya transgenji
Nkumuntu mushya 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 bishya bigomba guhindura uburyo bwo kubyitwaramo no kugenzura neza, kumenya neza, no kumenya imipaka ntarengwa mbere yo gukora ubushakashatsi bunini.
Intambwe ya 2:Tegura protocole yubushakashatsi
Igenzura ryiza: Koresha ADN isukuye irimo igice cyerekanwe nkicyitegererezo kugirango umenye niba sisitemu ya PCR nuburyo bisanzwe.
Igenzura ribi / ubusa: Koresha icyitegererezo cya ADN cyangwa ddH2O ibyo ntabwo bikubiyemo intego yibice 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.
Intambwe3: 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 amabwiriza hanyuma uyasome witonze, kandi witegure mbere yikigeragezo ukurikije amabwiriza.
Intambwe ya 4: Tegura sisitemu ya reaction ya PCR
Ukurikije protocole yubushakashatsi, vanga primers, H.2O, 2 × PCR ivanga, centrifuge hanyuma uyikwirakwize 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, ntabwo hakenewe uburyo bwo kweza aside nucleic iruhije.Icyitegererezo cyicyitegererezo gishobora gutegurwa muminota 10 hanyuma kongerwaho sisitemu ya PCR ihuye.
Icyitonderwa:
Uburyo bwa Lysis bufite ingaruka nziza zo gutahura, kandi ibicuruzwa byabonetse birashobora gukoreshwa mubitekerezo byinshi byo gutahura.
5.1: PCR itaziguye yamababi
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 bwa lysis uburyo
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 igomba kuba hagati ya 5-10% ya sisitemu ya PCR, kandi ntigomba kurenza 20% (urugero, muri sisitemu ya 20μl PCR, ongeramo 1-2μl ya lysis buffer, ntabwo irenze 4μl).
Intambwe ya 6: Igisubizo cya PCR
Nyuma ya centrifuging ya PCR reaction, shyira 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: Gutahura 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
Kugenzura ubuziranenge:
Ikizamini cyanga kugenzura ibintu bitandukanye byashyizwe 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 mu cyitegererezo.
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 cyerekana
Nyuma yubushakashatsi, koresha 2% sodium hypochlorite yumuti na 70% yumuti wa Ethanol kugirango uhanagure ahakorerwa ubushakashatsi kugirango wirinde kwanduza ibidukikije.
Umugereka
Imbonerahamwe 2. Ibisanzwe bikoreshwa muburyo bwa PCR bwo kumenya ibimera byahinduwe
Inyandiko yerekana:
SN / T 1202-2010, Uburyo bwiza bwa PCR bwo kumenya ibimera byahinduwe genetike mubiribwa.
Minisiteri y’ubuhinzi Itangazo 1485-5-2010, Gupima ibigize ibihingwa byahinduwe genetike nibicuruzwa byabo-umuceri M12 nibibukomokaho.
Igihe cyo kohereza: Jun-09-2021
