Qalabka Go'doominta Unugga ee RNA Wadarta Xirmooyinka Go'doominta ee unugga
Sharaxaada Qalabka:
Wadarta guud ee RNA oo si aad ah loo sifeeyay oo tayo sare leh ayaa laga heli karaa unugyo dhaqan oo kala duwan 11 daqiiqo gudaheed.
RNase-free
Ku shaqeeya heerkulka qolka (15-25 ℃)
Iyada oo leh Tiirka Nadiifinta DNA
Wax soo saarka RNA sare
Degdeg: Ku dhamee soo saarista 11 daqiiqo gudahood
Badbaadada: Ma jiro kiimiko dabiici ah
Sharaxaada
Xirmadani waxay isticmaashaa tiirka lafdhabarta iyo caanaha ay soo saartay Foregene, kaas oo si hufan uga soo saari kara nadiif-sare iyo wadarta tayada sare leh ee RNA unugyo lagu beeray 96, 24, 12, iyo 6-cells.
Qalabku wuxuu bixiyaa tiirka Nadiifinta DNA-ga oo hufan, kaas oo si fudud u kala saari kara supernatant iyo lysate unugga, xidhi kara oo ka saari kara DNA-da genomic.Qalliinku waa mid fudud oo waqti badbaadiya.
TIsagu RNA-kaliya tiirka ayaa si hufan ugu xidhi kara RNA oo leh qaacido gaar ah.Tiro badan oo muunado ah ayaa isku mar la tijaabin karaa.
Qaybaha kitka
| Halabuurka Kit | RE-03111 | RE-03114 |
| 50 T | 200 T | |
| kaydiye cRL1* | 25 ml | 100 ml |
| Buffer cRL2 | 15 ml | 60 ml |
| Bakhaar RW1* | 25 ml | 100 ml |
| Buffer RW2 | 24 ml | 96 ml |
| RNase-free ddH2O | 10 ml | 40 ml |
| Tiirka RNA-Kaliya | 50 | 200 |
| Tiirka-Nadiifinta DNA | 50 | 200 |
| Tilmaanta | 1 | 1 |
Fadlan xidho galoofyada oo qaado tillaabooyin ilaalin ah inta lagu jiro hawlgalka maadaama Buffer cRL1 iyo Buffer RW1 ay ku jiraan milixyo fowdo ah oo xanaaqa.
Tilmaamaha & faa'iidooyinka
■ Nidaamka oo dhan waxaa lagu shaqeeyaa heerkulka qolka (15-25 ℃), iyada oo aan baraf lagu qubeyn iyo heerkul hooseeya.
■ Qalabka oo dhan waa bilaash RNase, looma baahna in laga walwalo xaalufka RNA.
■ Tiirka Nadiifinta DNA-da ayaa si gaar ah u xiraya DNA-da, si ay xirmadu uga saarto wasakheynta DNA-da iyada oo aan lagu darin DNA-da dheeraad ah.
∎ Wax-soo-saarka RNA sare: Tiirka-RNA-kaliya iyo caanaha gaarka ah ayaa si hufan u nadiifin kara RNA.
■ Xawaaraha dheereeya: fududahay in lagu shaqeeyo waxaana lagu dhameyn karaa 11 daqiiqo.
■ Badbaadada: Looma baahna reageent organic.
■ Tayada sare: RNA-da la safeeyey waa nadiif sare, ka madhan borotiin iyo wasakh kale, waxayna la kulmi kartaa tijaabooyin kala duwan oo xiga.
Codsiga qalabka
Waxay ku habboon tahay soo saarista iyo nadiifinta wadarta RNA ee unugyada dhaqameed ee 96, 24, 12, iyo 6-cells.
Socodka shaqada
Jaantuska
Jaantuska baytariga agarose jel ee Qalabka Go'doominta unugga ee wadarta guud ee RNA wuxuu daaweeyay tirooyinka kala duwan ee kor ku xusan ee unugyada, 20μl mugga elution, qaado 2μl oo la safeeyey wadarta guud ee RNA 1%.
Kaydinta iyo nolosha shelf
Qalabka waxaa lagu kaydin karaa 12 bilood heerkulka qolka (15-25 ℃) ama 2-8 ℃ wakhti dheer (24 bilood).
Buffer cRL1 waxa lagu kaydin karaa 4 ℃ 1 bil kadib marka lagu daro 2-hydroxy-1-ethanethiol(ikhtiyaar).
RNA lama soo saarin ama wax soo saarka RNA waa yar yahay
Inta badan waxaa jira arrimo kala duwan oo saameeya waxtarka soo kabashada, sida: muunada unugyada RNA content, habka hawlgalka, mugga elution, iwm.
1. Barafka qubeyska ama cryogenic (4°C) centrifugation ayaa la sameeyay intii lagu jiray qaliinka.
Talo soo jeedin: Ku shaqee heerkulka qolka (15-25 ° C) inta lagu jiro habka oo dhan, ha ku maydhin baraf oo ha ku daboolin heerkul hoose.
2. Ilaalinta muunad khaldan ama wakhtiga kaydinta muunada xad dhaafka ah.
Talo-soo-jeedin: Ku kaydi shay-baarka--80 °C ama ku qabooji nitrogen dareeraha ah kana fogow isticmaalka baraf-dhalashada ee soo noqnoqda;isku day inaad u isticmaasho unug cusub ama unugyo dhaqan leh soo saarista RNA.
3. Shaybaar aan ku filnayn.
Talo soo jeedin: Marka unugyadu isku mid yihiin, hubi in unuggu si ku filan isu ekaysiiyo iyo in unugyadu si ku filan u kala qaybsamaan si loo sharaxo sii daynta RNA.
4. Eluent si sax ah looguma darin.
Talo soo jeedin: Xaqiiji in RNase-free ddH2O waxaa lagu daraa hoos udhaca dhexe ee xuubka tiirka sifaynta.
5. Mugga saxda ah ee ethanol buuxa laguma darin Buffer RL2 ama Buffer RW2.
Talo soo jeedin: Raac tilmaamaha, ku dar mugga saxda ah ee ethanol buuxa ee Buffer RL2 iyo Buffer RW2 oo si fiican isku qas ka hor intaadan isticmaalin qalabka.
6. Qiyaasta muunadda unugyada ma habboona.
Talo soo jeedin: Isticmaal 10-20 mg oo unug ah ama (1-5) × 106unug kasta 500 μl baffer RL1, maadaama isticmaalka nudaha xad-dhaafka ah ay keeni karto soo saarista RNA oo yaraada.
7. Mugga sare-u-qaadista oo aan sax ahayn ama muuqaal aan dhammaystirnayn.
Talo soo jeedin: Mugga sare ee tiirka nadiifinta waa 50-200 μl;Haddii saamaynta sare-u-qaadista aysan ku qanacsanayn, waxaa lagu talinayaa in la kordhiyo waqtiga meelaynta heerkulka qolka ka dib marka lagu daro RNase-free ddH preheated2O, tusaale ahaan 5-10 daqiiqo
8.Tiirka nadiifinta ayaa leh hadhaaga ethanol ka dib markii la dhaqo Buffer RW2.
Talo soo jeedin: Haddii ay jirto hadhaaga ethanol ka dib markii la dhaqo Buffer RW2, centrifugation tube madhan 1min, wakhtiga hawlgalka centrifugation tuubada madhan waxaa la kordhin karaa ilaa 2min, ama tiirka sifaynta waxaa la dhigi karaa heerkulka qolka 5 min si ku filan looga saaro ethanol hadhaaga ah.
RNA la safeeyey ayaa hoos u dhacay
Tayada RNA la safeeyey waxay la xiriirtaa arrimo ay ka mid yihiin ilaalinta shaybaarka, faddaraynta RNase, iyo khalkhalgelinta, iwm.
1. Tijaabada unugyada lama hayo wakhtigeeda.
Talo soo jeedin: Haddii shaybaarada unugyada ama unugyada aan loo isticmaalin waqti ku habboon ka dib ururinta, isla markiiba cryopreserve at -80 °C ama nitrogen dareere ah.Si aad u soo saarto RNA, isticmaal unug cusub oo la qaaday ama muunad unug mar kasta oo ay suurtogal tahay.
2. Baraf-dhalaalitaan soo noqnoqda ee shaybaarada unugyada.
Talo soo jeedin: Marka la kaydinayo shaybaarka unugyada, waxa fiican in la jarjaro qaybo yaryar si loo xafido, oo laga saaro mid ka mid ah qaybaha marka la isticmaalayo si looga fogaado baraf-dhalashada soo noqnoqda ee muunada iyo hoos u dhaca RNA.
3. RNase waa la soo bandhigay ama aan la xidhin galoofisyada la tuuri karo, waji-xidhka, iwm. inta lagu jiro qaliinka.
Talo soo jeedin: Tijaabooyinka soo saarista RNA waxaa sida ugu fiican loogu sameeyaa qolal kala duwan oo wax lagu sameeyo RNA miiskana waa la nadiifiyaa ka hor tijaabada.
Xiro galoofyada iyo maaskaro la tuuri karo inta lagu jiro tijaabada si loo yareeyo xaalufka RNA ee ay keento hordhaca RNase.
4. Reagen-yada waxa ku wasakhaysan RNase marka la isticmaalayo.
Talo-soo-jeedin: Ku beddel Xayawaanada cusub ee Wadarta Guud ee Go'doonka RNA ee tijaabooyinka la xidhiidha.
5. Tuubooyinka centrifuge, talooyinka, iwm. ee loo isticmaalo wax-ka-beddelka RNA waxay ku wasakhoobeen RNase.
Talo soo jeedin: Xaqiiji in tuubbooyinka centrifuge, talooyinka, pipettes, iwm. ee loo isticmaalo soo saarista RNA ay dhamaantood yihiin kuwo ka madax banaan RNase.
RNA la safeeyey ee la helay waxay saamaysaa tijaabooyinka hoose
RNA oo lagu nadiifiyo tiirka sifaynta, haddii ions milix ah, waxa ku jira borotiinku aad u weyn yahay waxay saamaynaysaa tijaabada hoose, sida: qoraalka dib u noqoshada, Northern Blot et al.
1. RNA-ga la macmalay waxa uu leeyahay hadhaaga ion cusbo.
Talo soo jeedin: Xaqiiji in mugga saxda ah ee ethanol lagu daray Buffer RW2 oo samee 2 tiir nadiifin ah oo dhaqida xawliga centrifugal ee lagu tilmaamay hawlgalka;Haddii ay jiraan wax hadhaaga ion cusbo, u dhaaf tiirka sifaynta Buffer RW2 5 daqiiqo heerkulka qolka oo samee centrifugation si aad u kordhiso ka saarida wasakhowga milixda.
2. Hadhaaga Ethanol ee RNA-ga la macmalay.
Talo soo jeedin: Xaqiiji in ka dib marka la dhaqo baffer RW2, samee hawlgalka centrifugation tuubada madhan xawaaraha centrifugation ee lagu tilmaamay hawlgalka, kordhiyo wakhtiga hawlgalka centrifugation tuubada madhan ilaa 2 min haddii ay weli jiraan hadhaa ethanol, ama ka tago heerkulka qolka 5 m
