The method of total yeast RNA extraction
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Last Update: 2021-01-24
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Source: Internet
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Author: User
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: What method does it take
extract the
RNA of the yeast? Do you have
reagents
boxes? What should I pay attention to for total yeast RNA extraction?
: the use of thermophenol method to extract yeast RNA, very good to use, so far has not failed.
(1) the yeast cells
cultured
in 3 ml selective
medium
, 30 degrees C overnight rotation culture.
(2) dilute the overnight cultured bacteria and re-culture the cells in a 10 ml medium.
(3) OD600 reaches 1.0 when the bacteria, 4000rpm/min centrifuge 5min, discard the culture base.
(4) suspends the cells in a 400 μl AE buffer (50mM Sodium Acetate, 10mM
EDTA
pH to 5.2).
(5) add 10% of the 1/10 volume
SDS
, fully mixed.
(6) to add an acidic phenol (pH4.5) with an equal volume preheated at 65 degrees C.
(7) 65 degrees C
heating
5min, 10min on ice.
(8) centrifuge 10min at room temperature 8000rpm/min.
(9) to take the water phase and add the same volume of phenol/chloroform/isoquinol (25:24:1), 10000rpm/min centrifugation 8min.
(10) to take the water phase and add iso-volume chloroform/isoquinol (49:1)10000rpm/min centrifugation 8min.
(11) to add 1/10 volume 3M pH5.2 sodium acetate, 2.5 times volume of pure ethanol, -20 degrees C overnight precipitation.
(12) precipitated sample at 4 degrees C centrifugation 5min, removing the liquid.
(13) dry RNA
ice
(place 15min) and finally dissolve the RNA with the appropriate amount of DECC water.
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