When performing an RNA extraction (to remove DNase for rt PCR) I shook the phenol chloroform before using. I thought the two organics were immiscible and needed mixing prior to use. Turns out I mixed the Phenol/chloroform phase with the equilibrating buffer phase which should not be used. I then centrifuges and took the upper aqueous phase. I thought something was wrong when the organic phase after centrifuging was only about 100 uL. So I am wondering if my RNA samples are ruined after being precipitated with that buffer phase. I took the upper phase, added 1/10 volume 3 M sodium acetate, then glycogen plus 3 volumes ice cold ethanol. So the original aqueous solution now contains the phenol chloroform isoamyl alcohol buffer. I am thinking I could salvage it by performing another phenol chloroform extraction. Is there anything in that buffer that will prevent use of this RNA?
At least for the phenol:chloroform:isoamyl alcohol from Sigma, the equilibration buffer is just TE (10 mM Tris, pH 8.0, 1 mM EDTA). That shouldn't affect your RNA, you just diluted it, but if it was precipitated afterwards, that shouldn't be a problem either.
That would be fantastic and I am hoping this is the case.
The phenol:chloroform:isoamyl alcohol only had about 100 mL left in a 400 mL bottle I believe. So the buffer constituted about half the solution and I just assumed it had to be mixed. It was not until I read the openwetware protocol notes on phenol:chloroform extraction about the buffer that I realized I made a mistake.
I added 200 uL of phenol:chloroform:isoamyl + TE buffer (hopefully). After centrifuging, there was about 100 uL organic phase, so there was an extra 100 uL in the aqueous phase, bringing the total to 300 uL. I believe I should need to add 300 uL more ethanol to facilitate precipitation.
Anyway i would use unbuffered acidic P/C/I for RNa extraction. Improves your yield.. And you have less DNA cause it stays in the interphase..
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