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Txheej txheem cej luam

Kev txheeb xyuas sai ntawm cov nroj tsuag transgenic

Text/Tong Yucheng

Kev sim ua haujlwm / Han Ying

Editor/Wen Youjun

Cov lus / 1600+

Lub sijhawm nyeem ntawv / 8-10 feeb

Kev txheeb xyuas sai ntawm cov nroj tsuag transgenic

Raws li tus neeg tuaj tshiab hauv chav kuaj, nws tsis yog txoj haujlwm zoo los tshuaj xyuas cov nroj tsuag zoo los ntawm ib pawg ntawm cov nroj tsuag uas tsis tshua muaj kev hloov pauv.Ua ntej, DNA yuav tsum tau muab rho tawm los ntawm ntau tus qauv ib los ntawm ib qho, thiab tom qab ntawd cov noob txawv teb chaws yuav raug kuaj los ntawm PCR.Txawm li cas los xij, cov txiaj ntsig feem ntau yog cov khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob..Puas yog nws tsis muaj txiaj ntsig zoo los ntsib cov txheej txheem sim thiab cov txiaj ntsig?Tsis txhob txhawj, kwv tij qhia koj yuav tshuaj xyuas cov nroj tsuag zoo transgenic yooj yim thiab raug.

Kauj ruam 1: Tsim kom pom primers

6.9-1

Txiav txim siab cov noob endogenous thiab exogenous gene kom kuaj tau raws li cov qauv yuav tsum tau kuaj, thiab xaiv tus neeg sawv cev 100-500bp ib ntus hauv cov noob rau primer tsim.Cov primers zoo tuaj yeem ua kom paub tseeb qhov tseeb ntawm cov txiaj ntsig tshawb pom thiab ua kom lub sijhawm tshawb pom luv luv (saib cov ntawv txuas ntxiv rau cov khoom siv feem ntau nrhiav pom).

Nco tseg:

Cov primers tsim tshiab yuav tsum tau ua kom zoo dua cov tshuaj tiv thaiv kab mob thiab txheeb xyuas qhov tseeb, qhov tseeb, thiab kev txwv tsis pub kuaj pom ua ntej ua qhov ntsuas loj.

Kauj ruam 2:Tsim cov txheej txheem kev sim

6.9-2

Kev tswj qhov zoo: Siv cov DNA ntshiab uas muaj lub hom phiaj tawg ua qauv los txiav txim seb PCR cov tshuaj tiv thaiv thiab cov xwm txheej zoo li cas.

Kev tswj tsis zoo / tsis pub dawb: Siv cov qauv DNA lossis ddH2O uas tsis muaj lub hom phiaj fragment raws li ib tug qauv los xyuas seb puas muaj ib qhov chaw ntawm cov kab mob nyob rau hauv PCR system.

Kev tswj xyuas sab hauv: siv cov primer / sojntsuam ua ke ntawm cov noob endogenous ntawm cov qauv los kuaj xyuas seb tus qauv puas tuaj yeem kuaj pom los ntawm PCR.

Nco tseg:

Kev tswj qhov zoo, qhov tsis zoo / qhov tsis zoo thiab kev tswj hwm sab hauv yuav tsum tau teeb tsa rau txhua qhov kev sim los ntsuas qhov siv tau ntawm cov txiaj ntsig kev sim.

Kauj ruam 3: Kev sim npaj

6.9-3

Ua ntej siv, saib seb cov tshuaj puas sib npaug.Yog tias pom nag lossis daus, nws yuav tsum tau yaj thiab sib tov raws li cov lus qhia ua ntej siv.2 × PCR mix yuav tsum tau pipetted thiab sib tov dua nrog ib tug micropipette ua ntej siv kom tsis txhob sib npaug ion faib.

Nco tseg:

Tshem tawm cov lus qhia thiab nyeem kom zoo, thiab npaj ua ntej kev sim ua raws li cov lus qhia nruj.

Kauj Ruam 4: Npaj PCR cov tshuaj tiv thaiv kab mob

6.9-4

Raws li kev sim raws tu qauv, sib tov cov primers, H2O, 2 × PCR sib tov, centrifuge thiab faib rau txhua lub raj tshuaj tiv thaiv.

Nco tseg:

Rau kev ntsuas loj lossis ntev ntev, nws raug nquahu kom siv PCR cov tshuaj tiv thaiv kab mob uas muaj UNG enzyme, uas tuaj yeem tiv thaiv aerosol paug los ntawm PCR cov khoom.

Kauj ruam 5: Ntxiv cov tshuaj tiv thaiv template

6.9-5

Siv Direct PCR thev naus laus zis, tsis tas yuav muaj cov txheej txheem purification nucleic acid txaus ntshai.Cov qauv qauv tuaj yeem npaj tsis pub dhau 10 feeb thiab ntxiv rau qhov sib thooj PCR cov tshuaj tiv thaiv.

Nco tseg:

Txoj kev Lysis muaj txiaj ntsig zoo rau kev tshawb nrhiav, thiab cov khoom tau txais tuaj yeem siv rau ntau qhov kev kuaj pom.

6.9-6

5.1: Direct PCR ntawm nplooj

Raws li qhov loj ntawm daim duab hauv phau ntawv qhia, txiav cov nplooj ntoo nrog lub cheeb ntawm 2-3mm thiab muab tso rau hauv PCR cov tshuaj tiv thaiv.

Nco tseg: Xyuas kom meej tias cov nplooj tawg tau muab tso rau hauv cov tshuaj tiv thaiv PCR, thiab tsis txhob ntxiv cov nplooj ntoo ntau dhau.

5.2: Leaf lysis method

Txiav cov nplooj ntawv nrog ib txoj kab uas hla ntawm 5-7 hli thiab muab tso rau hauv ib lub raj centrifuge.Yog tias koj xaiv cov nplooj paub tab, thov tsis txhob siv cov ntaub so ntswg ntawm cov hlab ntsha tseem ceeb ntawm nplooj.Pipette 50ul Buffer P1 lysate rau hauv lub raj centrifuge kom paub meej tias cov lysate tuaj yeem ua rau cov nplooj ntoo tag nrho, muab tso rau hauv lub thermal cycler lossis hlau da dej, thiab lyse ntawm 95 ° C rau 5-10 feeb.

6.9-7
6.9-8

Ntxiv 50ul Buffer P2 neutralization tov thiab sib tov zoo.Qhov tshwm sim lysate tuaj yeem siv los ua tus qauv thiab ntxiv rau PCR cov tshuaj tiv thaiv.

Nco tseg: Tus nqi ntawm cov qauv yuav tsum nyob nruab nrab ntawm 5-10% ntawm PCR system, thiab yuav tsum tsis txhob ntau tshaj 20% (piv txwv li, hauv 20μl PCR system, ntxiv 1-2μl ntawm lysis buffer, tsis ntau tshaj 4μl).

Kauj Ruam 6: PCR Reaction

6.9-9

Tom qab centrifuging lub PCR cov tshuaj tiv thaiv raj, muab tso rau hauv lub PCR ntsuas rau amplification.

Nco tseg:

Cov tshuaj tiv thaiv siv cov qauv tsis huv rau kev nthuav dav, yog li tus naj npawb ntawm cov voj voog amplification yog 5-10 ntau lub voj voog dua li thaum siv cov qauv DNA purified.

Kauj Ruam 7: Kev kuaj xyuas electrophoresis thiab kev txheeb xyuas qhov tshwm sim

6.9-10
6.9-11

 

 

 

 

 

 

 

 

 

 

 

M: 100bp DNA Ladder

1\4: Purified DNA txoj kev

2\5: Direct PCR txoj kev

3\6: Kev tswj tsis pub muaj

Kev Tswj Xyuas Zoo:

Cov txiaj ntsig ntawm kev sim ntawm ntau yam kev tswj hwm tau teeb tsa hauv qhov kev sim yuav tsum ua raws li cov xwm txheej hauv qab no.Txwv tsis pub, qhov laj thawj ntawm qhov teeb meem yuav tsum tau txheeb xyuas, thiab qhov kev sim yuav tsum tau ua dua tom qab qhov teeb meem raug tshem tawm.

Table 1. Cov txiaj ntsig zoo li qub ntawm ntau pawg tswj hwm

6.9-12

* Thaum cov plasmid siv los tswj qhov zoo, cov txiaj ntsig ntawm cov noob caj noob ces tuaj yeem ua rau tsis zoo

Kev txiav txim siab:

A. Cov txiaj ntsig ntawm cov noob endogenous ntawm cov qauv yog qhov tsis zoo, qhia tias DNA tsim nyog rau kev kuaj xyuas PCR zoo tib yam tsis tuaj yeem muab rho tawm los ntawm cov qauv lossis cov DNA rho tawm muaj cov tshuaj tiv thaiv PCR, thiab DNA yuav tsum tau muab rho tawm dua.

B. Cov txiaj ntsig ntawm cov noob endogenous ntawm cov qauv yog qhov zoo, thiab cov txiaj ntsig ntawm cov noob exogenous yog qhov tsis zoo, qhia tias DNA tsim nyog rau kev kuaj xyuas PCR zoo tib yam yog muab rho tawm los ntawm cov qauv, thiab nws tuaj yeem txiav txim siab tias XXX noob tsis raug kuaj pom hauv cov qauv.

C. Cov txiaj ntsig ntawm cov noob endogenous ntawm cov qauv yog qhov zoo, thiab cov txiaj ntsig ntawm cov noob exogenous yog qhov zoo, qhia tias DNA tsim nyog rau kev kuaj xyuas PCR zoo tib yam tau muab rho tawm los ntawm cov qauv, thiab cov qauv DNA muaj XXX gene.Kev kuaj pom tseeb tuaj yeem ua tau ntxiv.

Kauj Ruam 8: Tsim kom pom cov primers

 

6.9-13

Tom qab kev sim, siv 2% sodium hypochlorite tov thiab 70% ethanol tov los so qhov chaw sim kom tsis txhob muaj kuab paug ib puag ncig.

Cov ntawv ntxiv

Table 2. Feem ntau siv cov primers rau kev kuaj xyuas PCR ntawm cov nroj tsuag hloov caj ces

6.9-14

Cov ntaub ntawv siv:

SN/T 1202-2010, Qualitative PCR detection method for genetic modified plant ingredients in food.

Ministry of Agriculture tshaj tawm tsab xov xwm 1485-5-2010, Kev ntsuam xyuas cov khoom xyaw ntawm cov noob taum hloov cov nroj tsuag thiab lawv cov khoom-qib M12 thiab nws cov derivatives.


Post lub sij hawm: Jun-09-2021