
Qalabka baaritaanka Rnase (Fluorescence)
Xirmada ogaanshaha RNase waxay ku salaysan tahay baaraha RNA ee fluorophore-ku sumadeeyey.Marka muunadku aanu ku jirin dhaqdhaqaaqa RNase, baadhitaanku wuu xasiloon yahay mana soo saaro calaamada fluorescent;marka muunadku ka kooban yahay dhaqdhaqaaqa RNase, baadhida ayaa hoos u dhacaysa, taasoo keentay in si tartiib ah loo hagaajiyo calaamada fluorescence;heerka korodhka calaamada fluorescence waxay si togan ula xiriirtaa tirada iyo waxqabadka enzymes.Isticmaal akhristaha microplate fluorescence si aad u cabbirto hirarka dhererka ex/em=535/575nm si loo go'aamiyo in muunada uu wasakheeyay RNase.
Codsiga
Qalabkan waxa loo isticmaalaa in lagu ogaado sumowga RNase ee shaybaarada.
Cxoogsheegayaal
Magaca | HCP0035A-01 (192T) | HCP0035A-02 (48T) |
Xalka falcelinta 10× | 2.0ml | 0.5ml |
baaritaanka RNA | 1 tube | 1 tube |
TE baffer | 2.0ml | 0.5ml |
Heerka RNase A (10mg/ml) | 20μL | 10μL |
Halbeegga Dilution Buffer | 12ml | 6ml |
DNAse & Biyo aan sanka lahayn | 25ml | 25ml |
DNSase RNase ka fog | 50ml | 50ml |
Kaydinta iyo xasilloonida
1. Gaadiidka -25 ~ - 15 ℃;
2.Qaybaha kala duwan ee xirmada waxaa loo kaydiyaa si gooni gooni ah iyadoo loo eegayo heerkulka:
Magaca | heerkulka |
Xalka falcelinta 10× | -25 ~ - 15 ℃ |
baaritaanka RNA | -25 ~ - 15 ℃ |
TE baffer | -25 ~ - 15 ℃ |
Heerka RNase A (10mg/ml) | -25 ~ - 15 ℃ |
Halbeegga Dilution Buffer | -25 ~ - 15 ℃ |
DNAse & Biyo aan sanka lahayn | -25 ~ 30 ℃ |
DNSase RNase ka fog | 2 ~ 30℃ |
3. Ku kaydi xirmada aan la furin ilaa 12 bilood.
4.Ku kaydi xirmada 6 bilood ka dib markaad furto.Waxaa lagu talinayaa in la leexiyo xalka baaritaanka RNA iyadoo loo eegayo xaddiga isticmaalka kaliya si looga fogaado iftiinka iyo qaboojinta soo noqnoqda iyo dhalaalidda.
Qalabka iyo alaabta loo baahan yahay
1.Akhristaha florescence microplate (oo ay ku jiraan ex/em=535/575nm dhererka hirarka)
2. pipette-yada DNA-ga iyo RNase-la'aanta iyo talooyinka
3. Tuubbada EP ee DNA-da & RNase-la'aanta
4.DNase&RNase-free madaw oo saxan 96-ceel ah oo aan daahfurnayn
Diyaarinta reagent
1.Soo saar xirmada oo u dhig heerkulka qolka (18 ~ 25 ℃), rux oo isku qas qaybaha sida 10x falcelinta falcelinta, TE kaydinta, RNase A standard (10mg/mL), Dilution Buffer, ka dibna isla markiiba centrifuge.(Centrifuge at 4000 ~ 7000rpm ilaa 10 ilbiriqsi).
2. Centrifuge baaritaanka RNA at 4000 ~ 7000rpm 60 ilbiriqsi si aad ugu ururiso tuubada hoose, si taxadar leh u fur furka tuubada, oo ku dar 40μL TE baffer si ay u milmaan sida xalka kaydinta baaritaanka RNA, ka saar xalka kaydinta RNA sida waafaqsan. ilaa inta la isticmaalo oo kaliya ku kaydi -25 ~ -15 °C si aad uga fogaato qaboojinta iyo dhalaalidda soo noqnoqda.Soo saar xalka kaydinta baaritaanka mar kasta oo aad tijaabiso, ku qas 50 jeer TE kaydinta (tusaale, ku dar 490μL TE baffer 12μL RNA probe) sida RNA barista Xalka shaqaynaysa.Ku kaydi inta kale ee baaritaanka RNA ee shaqaynaysa at-25 ~ -15 °C si aad uga fogaato iftiinka iyo barafaynta soo noqnoqda iyo dhalaalidda.
Tallaabooyinka ogaanshaha
1. Tallaabo lagu dejiyo faa'iidada ku habboon ka hor imtixaanka ugu horreeya, si looga fogaado khatarta luminta dareenka ama calaamado xad-dhaaf ah.
1) cabbirada qalabka:
Saxanka rux 10 ~ 15s ka hor intaan la ogaan;
Dhererka hirarka xiisaha λEx=535nm;
Dhererka mowjadda sii deynta λEm=575nm;
Isticmaal shaqada faa'iidada tooska ah;
Heerkulka 37 ℃;
Habka dhamaadka.
U deji faa'iidada si otomaatig ah loo cabbiro haddii ay suurtagal tahay, beddelkeeda isticmaal meel faa'iido dhexdhexaad ah marka hore.
Fiiro gaar ah: habka dejinta qalabyada kala duwan ma aha mid joogto ah, fadlan kala tasho alaab-qeybiyaha si aad u hesho faahfaahin.
2) Dooro 2 ceel saxan 96-ceel ah, ku dar 10μL RNA shaybaarka xalinta shaqada iyo 10μL 10 × falcelinta ceel kasta;
3) Hal ceel ku dar 80μL oo ah DNase&RNase biyo ah, kuna dar 79μL oo ah DNase&RNase biyo ah iyo 1μL RNase A standard (100mg/mL) ceelka kale.
4) Saxanka dhig meel mugdi ah 37°C kadibna tijaabi 30 daqiiqo kadib.
5) Haddii loo dejiyo faa'iidada si loo cabbiro, Qiimaha Gain waxaa lagu soo bandhigi doonaa bar-beegtida aaladda faylka xogta, oo lagu tilmaamay G1.
6) Markaad isticmaalayso meel faa'iido dhexdhexaad ah bilawga, waa in la ogaadaa in: haddii qiimaha sare ee fluorescence uu ka sarreeyo xadka sare ee qalabka, qiimaha faa'iidada waa in si habboon loo dhimo;haddii qiimaha sare ee fluorescence uu ka hooseeyo xadka sare ee qalabka, qiimaha faa'iidada waa in si habboon loo kordhiyo;Ugu dambeyntii, qiimaha faa'iidada ku habboon ayaa la helay, oo lagu tilmaamay G2.
2. Siyo cabbiraadaha qalabka:
Saxanka rux 10 ~ 15s ka hor intaan la ogaan;
Dhererka hirarka xiisaha λEx=485nm;
Dhererka mowjadda sii deynta λEm=525nm;
U deji qiimaha faa'iidada G1 ama G2 helay tallaabada 1;
Heerkulka 37 ℃;
Haddii akhristaha microplate uu taageero habka dhaqdhaqaaqa, waxaa lagu talinayaa in la isticmaalo habka ogaanshaha dhaqdhaqaaqa, iyada oo u dhaxaysa 1 ilaa 1.5 daqiiqo, wadarta wakhtigana waa 30 daqiiqo.
3.Tusaalaha diyaarinta
Mugga muunada lagu taliyey waa 80μL.Haddii muunada la tijaabinayo uu ka yar yahay 80μL, ku mil 80μL oo leh DNA-ga iyo biyo aan Nase-ka lahayn.
Marka muunada la tijaabinayo ay ku jiraan walxo saameeya iftiinka fluorophore-ka (sida xalalka mugdiga ah, walxaha viscous-ka ah ee xoogga leh ama kuwa dusha sare leh), muunadda waa in lagu qasi karaa biyo aan lahayn DNase iyo RNase, laakiin fadlan ogow in hawlgalka qasitaanku saamayn doono. dareenka.Si muunadda la tijaabiyo oo ay ku jiraan xakameynta dhaqdhaqaaqa RNase (sida xalalka xoogga ionic ee sarreeya, pH <4 ama pH> 9 buffers, denaturants borotiinka, iwm.), natiijada cabbirku waa guud ahaan waxqabadka enzyme ee xalka saamiga, ma aha dhaqdhaqaaqa shakhsi ahaaneed ee enzyme.
Ku milid DNase I heerka (10mg/mL) oo leh Dilution Buffer Standard sida soo socota:
Maya | habka diyaarinta | Xoog-saarid |
1 | 2μL RNase Heerka A + 198μL Heerka Dilution Buffer | 0.1mg/ml |
2 | 2μL No. 1 muunad + 198μL Heerka Dilution Buffer | 1× 10-3mg/ml |
3 | 2μL No. 2 muunad + 198μL Heerka Dilution Buffer | 1× 10-5mg/ml |
4 | 2μL No. 3 muunad + 198μLS Badbaadiyaha Qafiifka ah ee caadiga ah | 1× 10-7mg/ml |
5 | 100μL No. 4 muunad + 100μL Heerka Dilution Buffer | 5× 10-8mg/ml |
Ku qas muunada lambar 5 10 jeer biyo aan lahayn DNase&RNase:
6 | 20μL No. 5 muunad + 180μL DNA-ga & biyaha aan RNase-ka lahayn | 5× 10-9mg/ml, 5pg/ml |
No. 6 muunad waxaa loo isticmaalaa sidii xakamayn togan;Biyaha aan DNAase&RNase-ka lahayn waxa loo isticmaalaa xakamayn taban.
4.Qaadashada iyo tijaabinta
1) Ku dar 10μL RNA xalka shaqada iyo 10μL 10x falcelinta saxan 96-ceel ah.Dooro 4 ceel si aad ugu darto xakamaynta taban iyo xakamaynta togan siday u kala horreeyaan, iyo ceelasha kale si aad ugu darto shaybaarada la tijaabinayo.Waxa muunad kasta ku jira 2 ceel oo badan, ceelkiiba 80μL;
2) Isla markiiba tijaabi oo akhri qiimaha calaamadda fluorescence RFU0 ee 0min.Ka dib marka la geliyo mugdiga 37 ℃ 30min, tijaabi oo akhri qiimaha calaamadda fluorescence RFU30 30min mar labaad.Haddii habka firfircoon la qaato, dhammaan calaamadaha fluorescence ee 0 ~ 30min waa la akhrin karaa.
Fasiraadda natiijooyinka imtixaanka
Haddi RFU30≥2×RFU0, waxaa loo arkaa in muunada la tijaabinayo uu wasakheeyey RNase.
Fiiro gaar ah: haddii muunada la tijaabinayo ay si xun u wasakhowday ama ay ku jiraan walxo faragelinaya, waxa dhici karta in RFU0 (muunad la tijaabinayo)> RFU0 (muunad la doonayo in la tijaabiyo) <2 ×RFU0 la tijaabiyey), taasoo keentay xukun diidmo been ah.Waqtigan xaadirka ah, muunadda la tijaabinayo waa in horay loogu qasi doonaa biyo aan lahayn DNase&RNase, ka dibna la tijaabiyaa.
Ogaanshaha tirada
Marka muunadda la tijaabinayo ay wasakhaysan tahay oo ay lagama maarmaan tahay in la qiimeeyo qiimaha fiirsiga ee RNase ee muunadda, waxa lagu go'aamin karaa hababka soo socda:
Ku qasi RNase A halbeeg (10mg/ml) oo leh Badbaadiyaha Caadiga ah sida soo socota:
Maya | Habka diyaarinta | xooga saarid |
1 | 2μL RNase Heer ah +198μL Heerka Dilution Buffer | 0.1mg/ml |
2 | 2μL No. 1 muunad + 198μL Heerka Dilution Buffer | 1×10-3mg/ml |
3 | 2μL No. 2 muunad + 198μL Heerka Dilution Buffer | 1×10-5mg/ml |
4 | 2μL No. 3 muunad + 198μL Heerka Dilution Buffer | 1×10-7mg/ml |
5 | 100μL No.4 muunad+100μL Heerka Dilution Buffer | 5×10-8mg/ml |
6 | 100μL No.5 muunad+100μL Heerka Dilution Buffer | 2.5×10-8mg/ml |
7 | 100μL No. 6 muunad+100μL Heerka Dilution Buffer | 1.25×10-8mg/ml |
8 | 100μL No. 7 muunad+100μL Heerka Dilution Buffer | 6.25×10-9mg/ml |
9 | 100μL No. 8 muunad+100μL Heerka Dilution Buffer | 3. 125×10-9mg/ml |
Dabadeed ku qas tirsiga 6 ~ No. 9 muunadaha 10 jeer ee DNA-ga iyo biyaha aan lahayn RNase:
10 | 20μL No.6 muunad+180μL DNA-ga iyo biyo aan sanka lahayn | 2.5×10-9mg/ml |
11 | 20μL No. 7 muunad+180μL DNA iyo biyo aan sanka lahayn | 1.25×10-9 mg/ml |
12 | 20μL No. 8 muunad+180μL DNA iyo biyo aan sanka lahayn | 6.25×10-10 mg/ml |
13 | 20μL No. 9 muunad+180μL DNA iyo biyo aan sanka lahayn | 3. 13×10-10 mg/ml |
No. 10 ~ No. 13 muunado waxaa loo isticmaalaa sida halbeegyada;DNA-ga iyo Biyo-la'aanta RNase sida muunada 0-fiirinta
Tijaabi muunada 0-fiirinta, halbeegyada iyo muunada wasakhaysan si wada jir ah iyadoo loo eegayo tillaabooyinka ogaanshaha si loo helo RFU0 iyo RFU30.
Xisaabi ∆RFU = RFU30-RFU0, qaado ∆RFU (0 fiirsashada) iyo ∆RFU (standard) sida isku xidhka iyo fiirsashada RNase ee heerka sida abcissa (0 fiirsashada waa 0), samee ku habboonaanta toosan, oo xisaabi isla'egta ku habboon y = faas + B, iyo isku xidhka isku xidhka r waa inuu noqdaa ≥ 0.99.Keen ∆RFU (muunad wasakhaysan) isla'egta sida y, xisaabi x, oo ku dhufo muunad ka hor qasitaan badan si aad u hesho qiyaasta fiirsashada ee muunada wasakhaysan.
Fiiro gaar ah: Isbeddelka calaamadda qalabka awgeed, waxa dhici karta in ∆RFU<0, wakhtigan, loo xisaabiyo ∆RFU=0.
Waxqabadka ogaanshaha
1. Xadka lagu ogaanayo: RNase A: 0.313pg/ml, ~ 1.56×10-9U/μL
2.Saxnaanta: Isku-dubbaridka isku-dhafka ee kala duwanaanshuhu ≤ 10%, Isku-dubbaridka kala duwanaanshaha ≤ 15%
Fiiro gaar ah
1. Qalliinka muunad-darrida muunadku waa in uu ahaado sida ugu dhakhsaha badan ee suurtogalka ah.Waqti aad u dheer ayaa saameyn doona saxnaanta tijaabada.
2. Halbeegyada kala duwan ee qalabka calaamadaynta fluorescent enzyme way kala duwan yihiin.Deji faa'iidada ku habboon ka hor imtixaanka koowaad.
3. Dhammaan reajentiyeyaasha waa in si buuxda loo ruxaa ka hor inta aan la isticmaalin.Marka lagu darayo muunado, shaybaarada lagu daray waa in lagu daraa gunta hoose ee saxanka calaamadda insaymiska si fiican looga fogaado in lagu daro qaybta sare ee gidaarka ceelka.Markaad ku darayso muunado, fiiro gaar ah u yeelo ha ku daadin ama ha dhalin xumbo.
4. Halbeegga xirmada waa RNase A, unuggeeda firfircoonna waxaa lagu qeexaa hal unug ka mid ah ensaymku wuxuu keenaa kororka nuugista 1.0 ee 260 nm marka khamiirka RNA lagu nadiifiyo 37 ° C iyo pH 5.0[1].Konton unug ayaa qiyaastii u dhigma 1 unug Kunitz[2].
5.Si looga fogaado faddaraynta RNase dibadda ah, DNase RNase fog ayaa lagu buufin karaa dusha miiska tijaabada, galoofyada iyo meelaha kale.5 daqiiqo ka dib, ku nadiifi tuwaalo waraaqo nadiif ah ka dibna samee hawlgallo tijaabo ah oo xiga.