Virus RNA extraction method

, isothionate extraction method (this is a detailed step to mention the avian influenza virus for reference)
1, take 200ul samples plus negative control plus positive control to add 1.5 ml sterilization eppendorf tube;
2, add 600ul isocyanate, then add control and sample, add 200ul chloroform, reverse mixing;
3, 13000rpm centrifugation 15min;
4, at the end of the 3rd step centrifugation, take the same multi-eppendorf tube, add 400ul at -20 degrees C pre-cooled isopropyl alcohol;
5, take the 3rd step centrifugation of the upper clear (must not absorb to the middle white layer, step 3 centrifugal end to take out, the tube should not tilt as far as possible) transferred to the fourth step of the preparation of the tube, mixed upside down;
6, 13000rpm centrifugation 15min, gently poured up, on the absorbent paper as far as possible dry liquid,
ethanol, ethanol, 60075%; Upside down several times to wash the remaining isopropyl alcohol;
8, 13000rpm centrifugation 15min, gently pour over the clear, on the absorbent paper as far as possible dry liquid;
9, 4000rpm centrifugation 10sec, the residual liquid of the pipe wall to the bottom, with a trace gun head to dry, room temperature
drying
2-3min (not excessive drying, prevent the next RNA insoluble);
10, add 20ulDEPE water (added to depc pure water high-pressure water is DEPE water), gently dissolved RNA. 2000 rpm centrifugation 5sec, ice storage standby (preferably within 2 hours to avoid RNA degradation).
, TRIzol LS extraction method
extracts are
serum
, blood, cell
culture
liquid, chicken embryo urine sac and other liquid viruses. Extract as far as possible in small numbers of people, to prevent the air RNA enzyme contamination. Everything used should also be RNA-free.
1, in a 1.5 ml eppendorf tube to add the virus original solution 500ul, then add TRIzol LS500ul, fully mixed, room temperature placed 10min;
2, add 200ul chloroform, tight centrifugal tube cover, force shock centrifugal tube (solution fully emulsified, into emulsion, phaseless phenomenon), room temperature placed 10min min (due to low chloroform boiling point, volatile, oscillating centrifugal tube may burst, be careful);
3, 4 degrees C, 13000r/min centrifugation 15min, take the upper liquid phase into another tube (cut off suction white intermediate phase);
4, add isopropyl alcohol, gently reverse centrifugal tube fully mixed liquid, room temperature placed 10min;
5, 4 degrees C, 13000r/min centrifugation 15min, (at first glance you will find that there seems to be nothing in the tube, and then a closer look, you will find a star-point white sediment on the wall near the bottom of the tube, That's it) use the gun carefully sucks away all the upper clearing;
6, 1 ml 75% ethanol wash again, 4 degrees C, 8000r/min centrifugation 10min, (then you will find that the tube is nothing, don't worry, some, but because the amount is too little invisible) with the gun carefully sucks all the upper clear, dry 5min in the ultra-clean table,
7, add the appropriate amount of DEPC treatment water. (If the material source is abundant, the amount of water added is the next RT total minus the amount of other
reagents
; if you want to save some use, look at it yourself and try not to add too much water);
8, it is recommended to do RT immediately. To be preserved, it can be frozen at -70 degrees C after adding ethanol at the last step and can be stored for one year, or about 1 month after adding DEPC water.
, Trizol method extraction method (this is the step to mention avian influenza virus, for reference)
Trizol is suitable for
tissues
or cultured bacteria in
humans, animals, plants,
microorganisms
, with sample sizes from tens of milligrams to several grams.
1, take the chicken embryo urine sac, add 5-10 times the volume of Trizol liquid, mix well;
2, room temperature for 5 minutes, and then add chloroform at a rate of 0.2 ml per 1 ml of Trizol liquid, cover the centrifugal tube, shake the centrifuge tube with your hands for 15 seconds;
3, take the upper water phase in a new centrifugal tube, according to each ml of Trizol liquid plus 0.5 ml of isopropyl alcohol ratio added isopropyl alcohol, room temperature for 10 minutes, 12000g centrifugation for 10 minutes;
4, discard the liquid, according to each ml Trizol liquid to add at least 1 ml of the proportion of 75% ethanol, mix well, 4 degrees C at 7500g centrifugation for 5 minutes;
5, repeat step 4;
6, carefully discard the liquid, then dry at room temperature for 5-10 minutes, be careful not to dry too much, otherwise it will reduce the solubility of RNA;
7, and then dissolve the RNA in water and leave for 10 minutes.
.
1, the entire operation should be wearing a
mask
and disposable
gloves
, and as far as possible at low temperature operation;
2, chloroform before the homogenizer can be stored at -70 degrees C for more than a month, RNA precipitation in 70% ethanol can be stored for one week, -20 degrees C for one year.