Mouse Genotyping Kit

Cat No: HCR2021A

Cov khoom no yog cov khoom siv tsim los rau kev txheeb xyuas sai ntawm nas genotypes, suav nrog DNA crude extraction thiab PCR amplification system.Cov khoom siv tuaj yeem siv rau PCR amplification ncaj qha los ntawm nas tail, pob ntseg, ntiv taw thiab lwm yam ntaub so ntswg tom qab yooj yim cleavage los ntawm Lysis Buffer thiab Proteinase k.Tsis muaj kev zom zaub mov thaum hmo ntuj, phenol-chloroform extraction lossis kem purification, uas yog qhov yooj yim thiab ua rau lub sij hawm siv cov kev sim.Cov khoom tsim nyog rau kev nthuav dav ntawm lub hom phiaj tawg mus txog 2kb thiab multiplex PCR cov tshuaj tiv thaiv nrog txog li 3 khub ntawm primers.2 × Mouse Tissue Direct PCR Mix muaj genetic engineered DNA polymerase, Mg²⁺, dNTPs thiab ib qho kev ua kom zoo tsis zoo los muab kev ua haujlwm siab ua haujlwm siab thiab inhibitor kam rau ua, kom PCR cov tshuaj tiv thaiv tuaj yeem nqa tawm los ntawm kev ntxiv cov qauv thiab primers thiab rehydrating cov khoom rau 1 ×.PCR cov khoom lag luam nthuav dav nrog cov khoom no muaj qhov tseem ceeb "A" hauv paus ntawm 3' kawg thiab tuaj yeem siv ncaj qha rau TA cloning tom qab purification.

Cheebtsam

Kev cia khoom

Cov khoom yuav tsum khaws cia ntawm -25 ~ -15 ℃ rau 2 xyoos.Tom qab thawing, Lysis Buffer tuaj yeem khaws cia ntawm 2 ~ 8 ℃ rau siv sijhawm luv luv, thiab sib tov zoo thaum siv.

Daim ntawv thov

Cov khoom no yog tsim rau nas knockout tsom, transgenic nrhiav pom, genotyping thiab hais txog.

Nta

1.Kev ua haujlwm yooj yim: tsis tas yuav rho tawm genomic DNA;

2.Daim ntawv thov dav: haum rau kev nthuav dav ncaj qha ntawm ntau cov ntaub so ntswg nas.

Cov lus qhia

1.Kev tso tawm ntawm genomic DNA

1) Kev npaj ntawm lysate

Cov ntaub so ntswg lysate tau npaj raws li tus naj npawb ntawm cov qauv nas yuav tsum tau lysed (cov ntaub so ntswg lysate yuav tsum tau npaj rau ntawm qhov chaw raws li qhov ntau npaum li cas thiab sib xyaw kom zoo rau kev siv), thiab qhov feem ntawm cov tshuaj reagents xav tau rau ib qho qauv yog raws li hauv qab no:

2) Qauv Npaj thiab Lysis

Pom zoo siv daim ntaub

Siv ib qho tsim nyog ntawm cov ntaub so ntswg kuaj hauv cov raj centrifuge huv, ntxiv 200μL ntawm cov ntaub so ntswg tshiab lysate rau txhua lub centrifuge raj, vortex thiab co, ces incubate ntawm 55 ℃ rau 30mins, thiab ces kub ntawm 98 ℃ rau 3mins.

3) centrifugal twj

Co lub lysate zoo thiab centrifuge ntawm 12,000 rpm rau 5mins.Lub supernatant tuaj yeem siv ua qauv rau PCR amplification.Yog tias tus qauv xav tau rau kev khaws cia, hloov lub supernatant mus rau lwm lub sterile centrifuge raj thiab khaws cia ntawm -20 ℃ rau 2 lub lis piam.

2.PCR Amplification

Tshem tawm 2 × Mouse Tissue Direct PCR Mix los ntawm -20 ℃ thiab thaw rau dej khov, sib tov upside down thiab npaj PCR cov tshuaj tiv thaiv raws li cov lus hauv qab no (ua haujlwm rau dej khov):

Nco tseg:

a) Cov nyiaj ntxiv yuav tsum tsis pub tshaj 1/10 ntawm lub cev, thiab yog tias ntxiv ntau dhau, PCR amplification yuav raug txwv.

Pom zoo PCR Cov Cai

Nco tseg:

a) Annealing kub: Nrog rau kev siv rau Tm tus nqi ntawm primer, nws raug nquahu kom teem lub annealing kub rau qhov me me Tm tus nqi ntawm primer +3 ~ 5 ℃.

Tej teeb meem thiab kev daws teeb meem

1.Tsis muaj phiajcim strips

1) Cov khoom lysis ntau dhau.Xaiv tus qauv tsim nyog tshaj plaws, feem ntau tsis ntau tshaj 1/10 ntawm qhov system;

2) Cov qauv loj dhau lawm.Dilute lysate 10 zaug thiab tom qab ntawd ua kom nrov nrov, lossis txo cov qauv me me thiab rov ua dua;

3) Cov qauv ntaub so ntswg tsis tshiab.Nws raug nquahu kom siv cov ntaub so ntswg tshiab;

4) Cov primer tsis zoo.Siv genomic DNA rau amplification los xyuas cov primer zoo thiab optimize primer tsim.

2.Tsis tshwj xeeb amplification

1) Lub annealing kub yog tsawg dhau thiab lub voj voog tus lej siab dhau lawm.Ua kom qhov kub thiab txias thiab txo cov voj voog;

2) Template concentration siab dhau.Txo tus nqi ntawm template los yog dilute lub template 10 zaug tom qab amplification;

3) Tsis zoo primer specification.Optimize lub primer tsim.

Sau ntawv

1.Txhawm rau kom tsis txhob kis kab mob sib kis ntawm cov qauv, ntau cov qauv ntsuas yuav tsum tau npaj, thiab cov cuab yeej saum npoo tuaj yeem raug ntxuav nrog 2% sodium hypochlorite tov lossis nucleic acid ntxuav tom qab txhua qhov kev kuaj yog xav tau rov siv dua.

2.Nws raug nquahu kom siv cov ntaub so ntswg tshiab nas, thiab cov qauv ntim yuav tsum tsis txhob loj dhau kom tsis txhob cuam tshuam cov txiaj ntsig amplification.

3.Lysis Buffer yuav tsum tsis txhob nquag khov-thaw, thiab tuaj yeem khaws cia ntawm 2 ~ 8 ℃ rau siv sijhawm luv luv.Yog tias khaws cia ntawm qhov kub qis, nag lossis daus tuaj yeem tshwm sim, thiab yuav tsum tau yaj tag nrho ua ntej siv.

4.PCR Mix yuav tsum tsis txhob nquag khov-thaw, thiab tuaj yeem khaws cia ntawm 4 ℃ rau kev siv sijhawm luv luv.

5.Cov khoom no tsuas yog siv rau kev tshawb nrhiav kev tshawb fawb thiab yuav tsum tsis txhob siv rau hauv kev kuaj mob lossis kev kho mob.