Rnase Inhibitor
Murine RNase inhibitor waa xuubka RNase dib-u-dhaqaale oo la muujiyey oo laga nadiifiyey E.coli.Waxay ku xidhan tahay RNase A, B ama C ee saamiga 1: 1 iyada oo loo marayo isku xidhid aan wadajir ahayn, taas oo ka hortagaysa dhaqdhaqaaqa saddexda enzymes iyo ka ilaalinta RNA ee hoos u dhaca.Si kastaba ha ahaatee, waxtar uma laha RNase 1, RNase T1, S1 Nuclease, RNase H ama RNase ka Aspergillus.Murine RNase inhibitor waxaa tijaabiyay RT-PCR, RT-qPCR iyo IVT mRNA, waxayna la jaanqaadi jirtay qoraalada kala duwan ee ganacsiga ee Reverse transcriptases, polymerases DNA iyo polymerases RNA.
Marka la barbardhigo bini'aadamka RNase inhibitors, murine RNase inhibitor kuma jiraan laba cysteinees kuwaas oo aad ugu nugul oksaydheynta taas oo keenta firfircoonida xakameynta.Taas oo ka dhigaysa mid xasiloon marka la eego xaddi yar oo DTT ah (in ka yar 1 mM).Habkani waxa uu ka dhigayaa mid ku haboon in loo isticmaalo falcelinta halka xaddi badan oo DTT ah ay lid ku tahay falcelinta (tusaale-waqtiga dhabta ah ee RT-PCR).
Acodsi
Alaabtan waxa si weyn loogu isticmaali karaa tijaabo kasta oo faragelinta RNase ay suurtogal tahay si looga fogaado xaalufka RNA, sida:
1.Isku-dubaridka xadhigga ugu horreeya ee cDNA, RT-PCR, RT-qPCR, iwm.
2.Waxay RNA ka ilaalisaa hoos u dhaca inta lagu jiro qoraalka/tarjumaada fayraska gudaha (tusaale, habka taranka fayraska ee vitro).
3.Joojinta dhaqdhaqaaqa RNase inta lagu jiro kala-soocidda RNA iyo nadiifinta.
Xaaladaha Kaydinta
Alaabta waxaa lagu kaydin karaa at -25～-15 ℃, shaqaynaysa 2 sano.
Kaydinta kaydinta
50 mM KCl, 20mM HEPES-KOH (pH 7.6, 25 ℃), 8 mM DTT iyo 50% glycerol.
Qeexitaanka unugga
Qadarka murine RNase inhibitor loo baahan yahay si loo joojiyo dhaqdhaqaaqa 5ng ee ribonuclease A 50% ayaa lagu qeexay hal unug (U).
Miisaanka molecular ee borotiinka
Miisaanka kelli ee murine RNase inhibitor waa 50 kDa.
Xakamaynta tayada
Exonuclease Hawsha:
40 U of murine RNase inhibitor oo leh 1 μg λ -Hind III dheefshiidka DNA at 37℃ 16 saacadood ma dhaliyo hoos u dhac sida lagu go'aamiyay agarose gel electrophoresis.
Waxqabadka Endonuclease:
40 U of murine RNase inhibitor oo leh 1μ g λ DNA at 37℃ 16 saacadood ma keenayso hoos u dhac sida ay go'aamisay agarose gel electrophoresis.
Naajin Hawsha:
40U ee murine RNase inhibitor oo leh 1μg pBR322 at 37℃ 16 saacadood ma keenayso hoos u dhac sida lagu go'aamiyay agarose gel electrophoresis.
RNase Hawsha:
40U ee murine RNase inhibitor oo leh 1.6μg MS2 RNA ee 4h at 37℃ ma keenayso hoos u dhac sida lagu go'aamiyay agarose gel electrophoresis.
E.coli DNA:
40 U of murine RNase inhibitor waxaa lagu baara joogitaanka E. coli genomic DNA iyadoo la isticmaalayo TaqMan qPCR oo leh qalabyo u gaar ah E. coli 16S rRNA goobta.Wasakhda E. coli genomic DNA waa ≤ 0.1 pg/40 U.
Notes
1. Oscillation ama walaaqin rabshadu waxay u horseedi doontaa firfircooni la'aan enzyme.
2. Heerkulka ugu wanaagsan ee kan xannibaaduhu wuxuu ahaa 25-55 ℃, waxaana la damiyay 65 ℃ iyo ka sarreeya.
3.Dhaqdhaqaaqyada RNase H, RNase 1 iyo RNase T1 laguma joojin murine RNase inhibitor.
4.Xakamaynta dhaqdhaqaaqa RNase waxaa laga helay pH ballaaran (pH 5-9 dhamaantood waxay ahaayeen kuwo firfircoon), waxqabadka ugu sarreeya ayaa lagu arkay pH 7-8.
5.Maadaama ribonucleases ay caadi ahaan hayaan dhaqdhaqaaqa hoos yimaada xaaladaha denateynta, taxadar waa in la qaataa si looga fogaado in la diido unugyada RNase Inhibitor kuwaas oo isku dhejiyay ribonuclease.Si looga hortago sii daynta ribonuclease firfircoon, heerkulka ka weyn 50 °C iyo uruurinta sare ee urea ama walxaha kale ee ilkaha waa in laga fogaadaa.
