Kev cais DNA los ntawm E. coli yog cov txheej txheem tseem ceeb hauv kev ua molecular biology. Kab lus no yuav taug kev tag nrho cov txheej txheem, muab cov kauj ruam cov ncauj lus thiab cov lus piav qhia, ua kom koj nkag siab txog cov txheej txheem kev tshawb fawb thiab cov txheej txheem ntawm cov txheej txheem. Txawm hais tias koj yog tus kws tshawb fawb raws caij nyoog lossis tus tshiab rau chav kuaj, cov lus qhia no yuav muaj txiaj ntsig zoo.
Npaj cov cell tso cai
Sau cov hauv E. Coli cell
Thawj kauj ruam hauv kev cais tawm DNA los ntawm E. coli cuam tshuam nrog kev sau cov kab mob hlwb. Qhov no feem ntau yuav tsum muaj kev loj hlob E. coli hauv qhov nruab nrab ua kua kom txog thaum nws nce mus txog lub logarithmic kev loj hlob theem. Lub sijhawm yog qhov tseem ceeb vim tias cov hlwb nyob rau theem no yog feem ntau yuav pab tau thiab tsis yooj yim, uas yuav ua rau siab dua DNA tawm los.
Resuspended cells nyob rau hauv ib tug tsim nyog ntas
Cov cell sau tau txais rov qab los ntawm qhov tsis tsim nyog. Ib qho kev xaiv ib qho yog ib qho tris - edta (te) tsis, uas pab tswj kev ncaj ncees ntawm DNA thaum lub sijhawm rho tawm. Tus ntas yeej pab tau ntau lub hom phiaj: nws tshem tawm cov ph, chelates cov chaw sib txawv uas muaj peev xwm ua lwm yam DNA, thiab muab qee qhov chaw ua kom zoo tshaj plaws rau kev ua kom zoo nkauj ntxiv.
Centrifugation rau pellet hlwb
● Cov tsis muaj rau centrifugation (ceev thiab sijhawm)
Tom qab resuspending lub hlwb, qhov kev ncua ntawv yog raug rau centrifugation rau pellet lub hlwb. Centrifugation ceev thiab lub sijhawm yog qhov tsis tseem ceeb. Feem ntau, centrifugation yog ua nyob ib ncig ntawm 4,000 - 6,000 g rau 10 - 15 feeb ntawm 4 ° C. Qhov no ua kom pom tseeb tias cov hlwb ua ib lub pellet nruj rau hauv qab ntawm cov raj ntawm cov raj hauv av.
● Tseem ceeb ntawm pelleting zoo
Cov pelleting kom zoo yog qhov tseem ceeb kom cais cov hlwb los ntawm qhov nruab nrab ntawm nruab nrab thiab lwm yam soluble cov. Ib qho zoo - Tsim pellet ua cov kauj ruam tom ntej yooj yim dua thiab muaj txiaj ntsig ntau, ua rau cov hlwb tsis tshua muaj tseeb thiab, yog li ntawd, siab tshaj DNA tawm los.
Tshem tawm ntawm supernatant
● Cov tswv yim kev tshem tawm supernatant
Thaum cov hlwb yog pelleted, tus supernatant (cov kua saum cov pellet) yuav tsum ua tib zoo tshem yam tsis muaj kev cuam tshuam lub cell pellet. Qhov no feem ntau ua tiav siv micropipette. Nws yog qhov tseem ceeb heev los ua cov kauj ruam no mediculously kom tsis txhob poob ib qho hlwb.
● Ua kom ntseeg tau tsawg ntawm cov pellet cell
Kev ua kom muaj kev poob qis ntawm cov pellet cell cuam tshuam nrog kev ua tib zoo saib thiab, yog tias tsim nyog, ntau cov puag ncig ntawm centrifugation thiab supernatant tshem tawm. Lub hom phiaj yog khaws kom ntau li ntau cov hlwb li sai tau hauv pellet rau qhov siab tshaj plaws DNA rov qab.
Txuas ntxiv ntawm NUCLEI LISH Kev daws
● Cov tib ua ke ntawm nuclei lysis daws
Lub nuclei lus daws feem ntau muaj xab npum (zoo li SDS), xws li TIS - HCl), thiab ib qho chelating tus neeg sawv cev (zoo li Edta). Cov xab nphua kev cuam tshuam cov hnab ntawv ntawm tes thiab nuclear hnab ntawv, tso cov ntsiab lus cellular, suav nrog DNA, rau hauv kev daws teeb meem.
Lub luag haujlwm hauv kev rhuav tshem phab ntsa
Lub nuclei lysis daws tsis tsuas yog lyses cell membrane tab sis kuj tau cuam tshuam nrog cov tshuaj thiab lub hnab nyiaj ntawm Nuclear kom tso tawm mus rau hauv kev daws teeb meem.
Resuspension ntawm cov hlwb
● Cov kav xa mus kom tsis txhob muaj DNA shearing
Thaum lub nuclei lysis muaj ntxiv, cov cell yuav tsum tau rov kho dua maj mam kom tsis txhob muaj DNA shearing. Shearing tuaj yeem tsoo cov DNA rau hauv cov ntawv thov me, uas tuaj yeem muaj teeb meem rau cov ntawv thov hauv qab uas yuav tsum muaj siab siab - cov dej khov kho - DNA.
● Ua kom tiav kev rov ua tiav
Ua kom tiav cov nyiaj rov qab kom txhua lub hlwb yog lysed uniformly, maximizing DNA rov qab. Qhov no tuaj yeem ua tiav los ntawm maj mam pipetting lossis vortexing txoj kev daws teeb meem ntawm qhov nrawm.
Txo rau Laiss hlwb
● Cov chaw kub kub rau kev tsim kom loj hlob
Cov xov tooj uas resuspended yog muab tsim rau ntawm qhov kub tshwj xeeb kom ntseeg tau tias yog lysis. Qhov no feem ntau ua tiav ntawm 37 ° C rau 55 ° C. Qhov ntsuas kub thiab ntsuas kub thiab lub sijhawm muaj feem sib txawv nyob ntawm cov txheej txheem thiab cov kev cai tshwj xeeb ntawm DNA kev rho tawm cov khoom siv.
● Ntev rau cov lysis
Lub sijhawm raug rau kev tsim kom loj hlob yog nyob nruab nrab ntawm 20 txog 30 feeb, tab sis nws tuaj yeem hloov kho raws li kev ua haujlwm ntawm cov lysis cell liysis pom. Cov loj hlob ntev yuav tsim nyog rau cov lysis ua tiav tab sis yuav tsum muaj kev sib npaug ntawm cov kev pheej hmoo ntawm DNA degradation.
Cua txias rau chav sov
● Qhov tseem ceeb ntawm cov cua txias zuj zus
Tom qab tsim kom loj hlob, Lysate yog maj txias rau chav sov. Cov khoom txias zuj zus yuav pab rau hauv DNA thiab txo qis kev pheej hmoo ntawm kev pheej hmoo ntawm thermal poob siab, uas tuaj yeem muaj peev xwm ua rau DNA.
● Los cuam tshuam rau DNA thiab cellular shris
Cua txias rau chav kub tso cai rau cov kab ke cellular rau nag lossis ua kom nws yooj yim cais cov DNA hauv cov kauj ruam tom ntej. Qhov no kuj tseem pab tswj cov kev ua si enzyme thiab ua kom yooj yim ntawm kev tshem tawm ntawm RNA los ntawm Rnase Kev Kho Mob.
Ntxiv rau Rnase daws teeb meem
● Lub home rnase hauv cov txheej txheem
Rnase kev daws teeb meem tau ntxiv rau degrade RNA, uas tuaj yeem ua lwm yam ua pa - purify nrog DNA thiab cuam tshuam nrog cov kev thov hauv qab. Rnase xaiv cov khawb RNA, tawm hauv DNA tsis tau.
Kev Tiv Thaiv L SNA kev tsis huv
Tiv thaiv kev ua kom tsis txhob muaj kev sib kis tsis muaj zog yog qhov tseem ceeb rau kev thov uas yuav tsum muaj DNA ntshiab, xws li PCR thiab ua ntu zus. Rnow kev kho mob kom ntseeg tau tias cov DN cais tawm yog pub dawb los ntawm cov nyom tsis muaj zog.
Purification ntawm DNA
Cov hau kev rau cais DNA los ntawm lwm cov khoom siv ntawm tes
Ob peb txoj kev tuaj yeem siv los ua kom DNA los ntawm Lysate. Cov no suav nrog phenol - chloroform extraction, ethhanol nag lossis nag lossis nag lossis dab tsi, thiab cov khoom lag luam DNA. Txhua txoj kev muaj nws txoj kev zoo thiab kev coj ua, nrog cov khoom lag luam ua lag luam yooj yim thiab cov txiaj ntsig zoo ib yam.
Kev txiav txim siab rau DNA purity
Purity yog qhov tseem ceeb tshaj plaws rau kev ua tiav ntawm cov ntawv thov hauv qab dej. Cov khoom siv coj ua lag luam, xws li lub xovtooj kev kho E. coli DNA)
Cia thiab tuav ntawm cais DNA
● Cov kev coj ua zoo tshaj plaws rau khaws cia DNA
Thaum ua kev limhiam, DNA yuav tsum tau muab cia rau hauv cov tsis haum, xws li Tuffer haum, ntawm - 20 ° C lossis - 80 ° C rau ntev - Term cia. Zam txhob khov - Txaj mus txog thaum lawv tuaj yeem ua rau DNA degradation.
Ua kom tuav tau kev ntseeg siab DNA rau cov ntawv thov hauv qab
Txhawm rau khaws cov DNA kev ncaj ncees, siv tsis muaj menyuam, nucaste - Dawb hlab thiab cov kev daws teeb meem. Qhov no ua kom pom tseeb tias cov DNA tseem tsis tau muaj thiab haum rau cov ntawv thov xws li cloning, ua ntu zus, thiab pCr.
TxogCov blukit
Jiangsu hadgene tsim nws lub hauv paus chaw (10,000㎡ GMP nroj tsuag thiab R & Dzhou, nyob hauv Shzhou, nyob rau hauv Suzhen lub nroog zoo nkauj, thiab Shanghai, txuas nws lub chaw tsim khoom lag luam. Lub tsev kawm North Carolina hauv Asmeskas tam sim no tab tom tsim kho, txuas ntxiv mus nws qhov chaw thoob ntiaj teb. HANGGE tau ua ib qho kev sib tw piav qhia kev txhim kho cov khoom siv tshuaj kho tes, nrog rau kev tshawb xyuas cov kua qaub, kev txhim kho kev txhim kho, thiab QC sim. Cov khoom lag luam xiav yog tsim rau kev tswj hwm zoo, ua kom muaj kev ua tiav ntawm kev kho mob cellular innovor.
Lub Sijhawm Post: 2024 - 09 - 05 14:47:03
