RNase kalma ce mai mahimmanci wacce ɗalibai da yawa waɗanda galibi ke gudanar da gwaje-gwajen hakar RNA ba sa son ji.Cike da makamai, RNA da a ƙarshe aka fitar da su tare da phenol da chloroform mai guba mai guba ya lalace.Ni ban yi sulhu ba!!!A yau, bari mu dubi asalin sanannen Rnase.

Ribonuclease (RNase), ko RNase, wani yanki ne na tsakiya wanda zai iya sanya RNA ruwa zuwa ƙananan kwayoyin halitta.RNase, a matsayin ƙaramin sunadaran ƙwayoyin cuta, ba a saba gani ba .Babban zafin jiki na al'ada da matsanancin matsa lamba haifuwar tururi da masu hana furotin ba za su iya kashe shi gaba ɗaya ba.Kwanciyar hankali na RNase yafi fitowa daga haɗin disulfide a cikin tsarin.Misali, RNase da ake amfani da shi na pancreas na bovine yana da amino acid 124 kawai, amma ya ƙunshi ginshiƙan disulfide 4.Sulfur bond da disulfide bond suna ba RNase tare da ingantaccen yanayin zafi.Bugu da ƙari, rnase yana da ɗan ƙaramin nauyin kwayoyin halitta kuma yana iya hanzarta dawo da yanayin sa na asali a lokuta da yawa.

Baya ga kasancewa da kwanciyar hankali sosai.RNases suna ko'ina a cikin dakin gwaje-gwaje .RNase shine tsarin kariya na halitta.Ga cell, exogenous RNA sau da yawa m.Idan aka kwatanta da exogenous DNA, exogenous RNA sau da yawa ya fi hatsari.An rubuta RNA cikin farin ciki kuma an fassara shi, don haka kusan dukkanin kwayoyin halitta sun sami RNases don kare kai daga mamayewar RNA.Don haka, ƙwayoyin ƙwayoyin cuta da aka noma a cikin dakin gwaje-gwaje kuma ku da kuke cire RNA kuna fitar da ƙamshin RNase.Ruwan jikin mutum (tsitsi, hawaye, da sauransu) yana ɗauke da adadi mai yawa na RNase, don haka kada ku yi kuka lokacin da RNA ta lalace.Yawan kuka, mafi muni da lalacewar RNA!!Sister Daiyu ba ta dace da cirewar RNA ba!

Bugu da ƙari, fata mai laushi kuma tana ɗauke da RNase da yawa, kuma alamomi, pipettes, kofofin firiji, da hannayen ƙofa waɗanda fata ta taɓa suna ɗauke da RNase.

Tare da yawan tashin hankali, bari mu kalli yadda ake mu'amala da RNases.

Abu na farko da kowa ke tunanin lokacin cire RNA shineDEPC(diethyl pyrocarbonate).DEPC galibi yana cire furotin ta hanyar haɗawa da zoben imidazole na ƙungiyar RNase mai aiki histidine, ta haka yana hana ayyukan enzyme.0.1% DEPC na iya samun sakamako mai kyau na cirewa akan Rnase, amma muna buƙatar kula da cewa DEPC sanannen carcinogen ne, don haka ya kamata a biya kulawa ta musamman lokacin amfani da shi.

Don RNase, muna buƙatar farawa daga bangarori biyu,na farko shine hana ayyukan RNase na ciki

Reagents na RNA na al'ada kamar guanidine isothiocyanate da DTT da ke cikin Trizol na iya buɗe haɗin disulfide na RNase, amma har yanzu akwai wasu RNases, musamman a samfuran nama, don haka kula da ƙarancin zafin jiki.

1.Nan da nan nutsar da samfurin nama a cikin ruwa nitrogen bayan fitar da shi, ko a cikin maganin adana RNA na kasuwanci.

2.Bayan cire RNA na samfurin tantanin halitta, ƙara shi zuwa maganin lysis kuma a yi shi a kan akwatin kankara

3. Zai fi kyau a yi amfani da nitrogen na ruwa don niƙa lokacin da samfurin nama ya zama homogenized.Lokacin amfani da homogenizer na lantarki ba tare da nitrogen mai ruwa ba, kula da cikakken pre-sanyi adaftar homogenate.

Na biyu shine exogenous DNAse

1.Kasance da cikakken makami, sa rigar lab, sanya abin rufe fuska, kuma a tabbata kun sanya sabbin safar hannu guda biyu (kada ku kasance masu kishi sosai! Ya kamata a lura cewa Trizol yana da lalata sosai, kuma yana da ƙarfi ta hanyar safar hannu, don haka kada ku ɗigo a hannunku).

2.Duk shawarwarin pipette da aka yi amfani da su, bututun EP, bututun PCR da sauran kayan aikin dole ne a yi amfani da su de-RNase.Ana iya jiƙa shi a cikin 0.1% DEPC sannan a matsa shi a ƙarƙashin babban matsin lamba.Kula da aiki a cikin murfin hayaki.Azzalumai na gida na iya siyan kayan amfani kai tsaye don cire enzymes.

PS: Bari in gaya muku wata kasala hanya.Kodayake yawan zafin jiki da matsa lamba ba za su iya cire RNase gaba ɗaya ba, zai cire babban ɓangaren sa.Sau 2 na yawan zafin jiki da matsa lamba yana da tasiri mai kyau, kuma cirewar RNA yana da ɗan tasiri.

3.Alcohol na iya lalata furotin,don haka ana iya goge teburin cirewar RNA da barasa 75%. , kuma ana iya fesa safar hannu da barasa.

4.Maganin narkar da RNA na ƙarshe da bututun centrifuge shima yakamata a yi maganin de-RNase.Ruwan DEPC shine maganin narkar da RNA da aka saba amfani dashi.Bari muyi magana game da hanyar shiri daidai na ruwan DEPC (tuna don sake dawo da DEPC na kasuwanci lokacin da kuka siya)

Ƙara DEPC zuwa ruwa mai zafi a 1: 1000, girgiza da kyau, bari a tsaya a cikin dare a 37 ° C, kuma bakara a 121 ° C a ƙarƙashin babban zafin jiki da matsa lamba na minti 15.Ana iya adana ruwan DEPC a -20 ° C a cikin 1ml aliquots.

A ƙarshe, don taƙaitawa: ƙananan zafin jiki shine maɓalli, kayan amfani ana sarrafa su da kyau, cikakkun makamai da ƙarancin magana!

To, shi ke nan don dabarun yau.Ina yi muku fatan duk abubuwan da ke tattare da RNA da tsabta da kuka ambata.Dukansu A260/A280 sune 2.0!!!

Hakika, idan kun yi amfani da akayan aikin hakar RNA zafin ɗaki, ƙila ba za ku ci karo da matsalolin da ke sama ba.

Aiki a zafin jiki, ba tare da ƙara DNA ba, yana fitar da jimillar RNA daga sel a cikin mintuna 11, kuma yana fitar da jimillar RNA daga kyallen dabba ko tsire-tsire a cikin mintuna 30.

Don samfuran gwaji, tuntuɓi:overseas@foregene.com

Samfura masu dangantaka:

https://www.foreivd.com/cell-total-rna-isolation-kit-product/

https://www.foreivd.com/animal-total-rna-isolation-kit-product/

https://www.foreivd.com/plant-total-rna/