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RNA extraction by trizol


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3 replies to this topic

#1 Roshan

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Posted 16 May 2013 - 12:13 PM

Hey guyz,
If someone is doing RNA extraction by Trizol method Can the the protein pellet that is obtained be used for expression analysis or this method waste the cellular proteins. I wanted the protein pellet for doing SDSPAGE and 2D

Regards,
Roshan

#2 Curtis

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Posted 16 May 2013 - 07:04 PM

According to which protocol ?

#3 Roshan

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Posted 17 May 2013 - 12:04 AM

We use manufacturers protocol Currently we are using invitrogen trizol method

The steps are below
Tissue sample+500ul Trizol
Homogenize
incubate at 5' room temp
0.2 ml chloroform
mix by inversion for 15sec
Incubate at room temp for 15'
Centrifuge at 12, 000g for 15'
three layers are formed The pellet iz protein
aqueous layer separate
add isopropanol
incubate at room temp for 10'
centrifuge at 12000g for 10'
Decant teh supernatant
wash pellet with ethanol
and briefly vorex
centrifuge for 5'
remove ethanol air dry and redissolve the RNA in DEPC



Roshan

#4 Curtis

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Posted 17 May 2013 - 09:32 AM

There is no pellet at that step. I do this every week, unless you mean the interphase.




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