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

Ishning borishi. 10 g yog`sizlantirilgan paxta chigitining kukunini standart tuzli eritma (STE) bilan shovonchada gomogen sholatga keltiriladi, bunda eritmaning qovushqoqligi ortadi. Gomogenatga teng shajmda suvga to`yintirilgan fenol qo`shiladi va aralashmani 30-40 minut davomida chayqatilib, keyin sentrifugada 15 min aylantiriladi. Bunda 3 ta qatlam shosil bo`ladi: pastki qatlam – denaturatsiyaga uchragan oqsil cho`kmasi, o`rta qatlam – fenol va yuqori qatlam suvli qatlamdan iborat. Fenol va suv qatlamlari oraligida denaturatsiyaga uchragan oqsilning yupqa qavati shosil bo`ladi. Suv qismida DNK bo`lib, uni pipetka yordamida tortib olinadi. Suv qismidan fenolni yo`qotish uchun efir bilan ishlanadi (1 shajmda ikki marotaba, ammo bu bosqichni qisqartirish mumkin). Suv qismidagi DNK-ni 2,5-3 shajmdagi sovutilgan 96-li spirt yordamida cho`ktiriladi, buning uchun oldinroq DNK eritmasining ion kuchini 1 M-gacha keltiriladi (5 M nariy atsetat yordamida). DNK eritmasini oz-oz miqdorda spirtga qo`shiladi. DNK ipsimon cho`kma sholida eritma yuzasiga chiqadi. DNK iplarini 20 ml standart tuzi eritmada eritiladi va unga 1 M-gacha natriy atsetatdan qo`shiladi va spirt bilan cho`ktirish qaytariladi. DNK-ni shisha tayoqchaga o`raladi va uni filtr qog`ozi orasiga olib siqiladi va 20-30 ml STE-da eritiladi. eritmaning 260 nm-da optik zichligi o`lchanadi va DNK-ning chiqish miqdori aniqlanadi (A₂₆₀ dagi 1 optik birlik 47 mkg DNK-ga teng). A₂₆₀A₂₈₀ va A₂₆₀A₂₃₀ nisbatlarni aniqlanadi. Birinchi nisbat qoldiq oqsil mikdorini (oqsildan tozalangan DNK2,00), ikkinchi nisbat qoldiq polisaxaridlarning miqdorini aniqlaydi.
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