Guruh talabasi ning biorganik kimyo fanidan amaliy mashg’uloti namangan 2023

2-Mavzu: Nuklein kislotalar (RNK, DNK) ni tabiiy manbalar(paxta chigiti, undirilgan mosh, loviya, fasol, no`xat) dan ajratib olish
2-ish
Undirilgan loviyadan umumiy RNK ajratib olish
Ishning umumiy izoshi. Fenol – detergent yordamida loviya kurtagidan umumiy RNK ajratib olinib, uning tozalik darajasi va unumini aniqlash.
Jihozlar va reaktivlar.
TSentrifuga, shovoncha, aralashtirgich, 50 ml-li o`lchov silindri, 10 ml-li pipetka yoki 10-20 ml-li shpritslar, standart tuzli eritma – 0,14 M Na(K)-sitrat, 200 ml etil spirti, suvga to`yingan fenol, pH 6,0-li 10%-li Na-dodetsilsulfat (Na-DDS), xloroform, izoamil spirti, 5 g ungan loviya.
Ishning borishi. Loviyaning unib chiqqan qismini vodoprovod suvi bilan va keyin distilangan suv bilan yaxshilab yuviladi. Undan 5 g olib, chini shovonchada 50 ml standart tuzi eritmada gomogen sholatga keltirililadi va gomogenatni 3-4 qavatli dokadan o`tkaziladi. Bu gomogenatga 1%-gacha Na-dodetsilsulfat qo`shiladi va aralashtiriladi va uning ko`pirib ketmasligiga e`tibor berib, suvga to`yintirilgan pH 6,0-li fenol qo`shiladi. Aralashmani 60 C-gacha qizdiriladi va 30 minut davomida yaxshilab chayqatiladi, 0 C -  5 C atrofida sovutiladi va 15-20 minut davomida sentrifugalanadi. Bunda 3 ta qavat shosil bo`ladi: suvli, fenol va cho`kma. Suvli qatlam deproteinlashga uchragan RNK-dan tarkib topgan, uni shprits yordamida ajratib olinadi va teng shamdagi xloroform va izoamil spirti aralashmasi (24:1) bilan deproteinlanadi. Aralashmani fenol bilan ishlanganidek yaxshlab chayqatiladi, sentrifuga qilinadi va yuqoridagi suvli qavat ajratib olinadi. RNK suvli qavatdan 2,5 shamjdagi spirt bilan cho`ktiriladi (–10 S). TSentrifuga yordamida cho`kmani ajratib olinadi va 10-20 ml standart tuzi eritmada eritiladi yoki boshqa bufer eritma ishlatish mumkin va 260, 280 va 230 nm-da yutilish qiymatlari aniqlanadi. A<sub>260</sub>A<sub>230</sub> va A<sub>260</sub>A<sub>280</sub> aniqlanadi. Birinchi nisbat RNK-ning polisaxaridlardan, ikkinchisi oqsillardan tozalanish darajasini belgilaydi (2,00). 260 nm-dagi yutilish yordamida RNK kontsentratsiyasi va shosil bo`lgan miqdori aniqlanadi (260 nm-dagi 1 optik birlik qiymatga 42 mkg RNK to`g`ri keladi).
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