Kusaka kwa DNA kuchokera ku E. Coli ndi njira yofunika kwambiri mu biology subilogy. Nkhaniyi idzakuyendetsani ntchito yonseyo, ndikupereka masitepe ndi malongosoledwe, onetsetsani kuti mumvetsetsa za sayansi ndi mbali zothandiza pazinthu. Kaya ndinu wofufuza kapena watsopano ku labu, bukuli lidzakhala chinthu chamtengo wapatali.
Kukonzekera kwa kuyimitsidwa kwa cell
● Kutengera kwa E. Coli
Gawo loyamba pakupanga DNA kuchokera ku E. Coliri imaphatikizapo kusonkhanitsa mabakiteriya. Izi nthawi zambiri zimafuna kukula e. coli mu madzi abwino kwambiri mpaka ikafika gawo lagalasi. Nthawiyo ndi yofunikira chifukwa maselo mu gawo lino amakhala othandiza kwambiri komanso osavuta kutuluka, zomwe zimabweretsa zokolola zapamwamba.
● Kuthetsa maselo obwezeretsanso mu buffer yoyenera
Maselo omwe amasonkhanitsidwa amathetsedwa mu buffer yoyenera. Chosankha chodziwika bwino ndi Tris - EDTA (Te) Buffer, chomwe chimathandiza kusungabe kukhulupirika kwa DNA pakuwunika. Buffer imakwaniritsa zifukwa zingapo: zimakhazikika pa PH, Chelates Cirs Cipoti omwe angatsimikizire DNA, ndipo amapereka malo oyenera a ionic kuti pambuyo pake.
Centrifugation ku ma cell a pellet
● magawo a centrifugation (kuthamanga ndi nthawi)
Pambuyo pakuthetsa maselo, kuyimitsidwa kumaperekedwa kwa centrifigation ku Pellet maselo. Kuthamanga kwa Centrifugation ndi nthawi yovuta kwambiri. Nthawi zambiri, centrifugation imachitika pafupifupi 4,000 - 6,000 g kwa 10 - mphindi 15 pa 4 ° C. Izi zikuwonetsetsa kuti maselo amapanga pellet wolimba pansi pa centrifuge chubu.
● Kufunika koyenera
Zosankha zoyenera ndizofunikira kuti mulekanitse maselo kuchokera mu sing'anga kapena zigawo zina zosungunulira. Chitsime - Cholembera chopangidwa chimapangitsa kuti zinthu zikhale zosavuta komanso zothandiza, zoyambitsa zopeputsa zochepa za DNA zokolola za DNA.
Kuchotsa ndalama
● Maluso pazochotsa ndalama
Maselo akangopendekeka, sublenatant yayikulu (madzi pamwamba pa pellet) ayenera kuchotsedwa mosamala popanda kusokoneza pellet pellet. Izi nthawi zambiri zimachitika pogwiritsa ntchito tizilombo. Ndikofunikira kuchita izi mosamalitsa kupewa kutaya maselo aliwonse.
● Kuyambitsa kuchepa kochepa kwa khungu la cell
Kuwonetsetsa kuti kuchepa kwa telet yam'manja kumaphatikizapo kusamala mosamala ndipo ngati kuli koyenera, kuzungulira kwa magawo angapo a centrifugation ndi kuchotsa ndalama. Cholinga ndikusunga maselo ambiri momwe mungathere mu pellet pakuchira kwakukulu kwa DNA.
Zowonjezera za Social Nyimbo Yabwino
● Zigawo zikuluzikulu za nyukiliya
The Neclei Lysis nthawi zambiri imakhala ndi zotchinga (monga SDS), buffer (monga tris - HCL), komanso Edta). Wotchipayo amasokoneza cell nembanemba ndi emvulopu ya nyukiliya, osayiketsa, kuphatikizapo DNA, mu yankho.
● Udindo wophwanya makoma a cell
Kusasinthika kwa mafilimu sikungongobweretsa nembanemba komanso mapika, ndikuphwanya makhoma a cell ndi maenvelopu kuti atulutse DNA mu yankho.
Kukhazikika kwa maselo
● Kupaka mapaupi odekha kuti mupewe kumeta kwa DNA
Nthawi yomweyo fumbi la nuclei likawonjezeredwa, maselo amafunikira kuti athetsedwe modekha kuti apewe kumeta kwa DNA. Kumeta kumatha kuswa DNA ku zidutswa zazing'ono, zomwe zimatha kuvutitsa pakugwiritsa ntchito kwambiri - molecular - kulemera kwa DNA.
● Kuonetsetsa kuti mudzakwanitsa
Kukhazikika kwathunthu kumatsimikizira kuti maselo onse amaswedwa mosafanana, kukulitsa kuchira kwa DNA. Izi zitha kuchitika chifukwa cha mapaipi odekha kapena ogwiritsa ntchito njira yothetsera pang'ono.
Makulitsidwe ku ma cell a Lyse
● Kukhazikika kwa kutentha kwa makulitsidwe
Maselo omwe athedwa amakhala okhazikika pamtunda wapadera kuti atsimikizire ma lysis athunthu. Izi nthawi zambiri zimachitika pa 37 ° C mpaka 55 ° C. Kutentha kwenikweni ndi nthawi yayitali kumatha kusiyanasiyana malinga ndi protocol ndi zofunikira zina za DNA iso.
● Kutalika kumafunikira lysis yothandiza
Kutalika kwa makulidwe kuli pakati pa mphindi 20 mpaka 30, koma kumatha kusinthidwa malinga ndi luso la ma cell lysis. Kutalika kwa nthawi yayitali kungakhale kofunikira kumaliza ma lysis koma ayenera kukhala okhazikika pachiwopsezo cha DNA kuwonongeka kwa DNA.
Kuzizira kutentha kwa chipinda
● Kufunika kwa nthawi yozizira
Pambuyo makulitsidwe, mawu akhungu amakhazikika pang'onopang'ono mpaka kutentha. Kuzizira pang'onopang'ono kumathandizira kukhazikika kwa DNA ndikuchepetsa chiopsezo cha kugwedezeka kwa mafuta, komwe kumatha kunyoza DNA.
● Zotsatira za DNA ndi zinyalala
Kuzizira kutentha kwa chipinda kumalola zinyalala zam'manja pamtunda, zimapangitsa kuti zikhale zosavuta kulekanitsa DNA pamayendedwe otsatizana. Izi zimathandizanso kukhazikika kwa enzyme ntchito ndikuthandizira kuchotsedwa kwa RNA mwa chithandizo cha rnase.
Kuwonjezera kwa njira ya rnase
● Cholinga cha ronase munjira
RANASE yankho limawonjezeredwa kuti muchepetse RNA, yomwe ingakhale ngati co - Choyeretsa ndi DNA ndikusokoneza ntchito zotsika. Ruase mosankha ma RNA, kusiya DNA yolimba.
● Kuletsa kuipitsidwa kwa RNA
Kupewa kuipitsidwa kwa RNA ndikofunikira pazogwiritsa ntchito zomwe zimafunikira DNA yoyera, monga PCR ndi sequening. Chithandizo cha rtase chimatsimikizira kuti DNA yadzitali ndi ya RNA yoyipitsa.
Kuyeretsa DNA
● Njira zolekanitsa DNA kuchokera pazida zina zam'manja
Njira zingapo zitha kugwiritsidwa ntchito kuyeretsa DNA kuchokera ku Lysate. Izi zikuphatikizanso phenol - Chlorotorm zowonjezera, ethanol mpweya, ndikugwiritsa ntchito malonda E. Coli DNA Makiland. Njira iliyonse ili ndi zabwino zake komanso zipwirikiti, ndi zamalonda zopereka zothandizira komanso zotsatira zosasinthasintha.
● Maganizo a DNA kuyera kwa DNA
Kuyera ndi chinthu chofunikira kwambiri kuti chipambane pazinthu zotsika. Makina ogulitsa, monga mankhwala ochiritsira a cell E. Coli DNA, adapangidwa kuti atulutse - kuyera kwa DNA pochotsa mapuloteni, lipids, ndi zodetsa zina.
Kusunga ndikugwira ntchito kwa DNA
● Zochita zabwino zosungira DNA
Kamodzinso kuyeretsedwa, DNA iyenera kusungidwa mu buffer yoyenera, monga terfder, pa - 20 - 80 - 80 c kwa nthawi yayitali - Kusunga kwa mawu. Pewani Kupuma Kwambiri - Thaw Miycles momwe iwo angapangire kuwonongeka kwa DNA.
● Kusungabe kukhulupirika kwa DNA pakugwiritsa ntchito
Kuti musunge DNA Kukhulupirika, gwiritsani ntchito osabala, nuclese - machubu aulere ndi mayankho. Izi zikuwonetsetsa kuti DNA idasalamulidwa komanso yoyenera kugwiritsa ntchito ntchito monga kusungira, kupindika, ndi pcr.
ZaBuluuni
Jiangsu Hillgene anakhazikitsa likulu lake (10,000 gmp likulu la R & D) ku Suzhou, malo awiri opanga a Suzhou, malo opanga nyanja ku Shenzhen ndi Shanghai! Tsamba la North Carolina ku US lili pangozi yomwe ikumangidwa pano ikufalikira padziko lonse lapansi. Mapiri akwera njira yofotokozera zinthu zothandizirana ndi ma cell, kuti apezeke, ndi nsanja - Kuyimitsidwa kwaulere Kukulalikira, Kuyesedwa Kwabwino, ndi Kuyesa QC. Zogulitsa za buluu zimapangidwa kuti ziziwongolera bwino, kuonetsetsa kupambana kwa ma cell othandizanso.
Post Nthawi: 2024 - 09 - 05 14:47:03
