3 replies to this topic

[dkkorzow]

I am attempting to extract DNA from some sediment samples.  We know that they are low biomass.  I did a phenol/chloroform extraction and then an isopropanol precipitation.  I have CTAB and SDS in the extraction solution.  I put in carrier before the precipitation because I know the DNA concentration may be very low.  I added 1 volume isoprop and then let sit at 4C for 30 min and the centrifuged at room temp max speed for 20 min.  After that I did not have a pellet, but a clear viscus drop of liquid at the bottom of the tube.  That drop contained the carrier (glycol blue so I could see the color to know it was there).  The drop was only about 15-20ul, so I know I didn't take the wrong phase from the chloroform extraction and I was very, very careful to not have organic carry over so I do not think it is that.  Also, it appears in the blanks as well as the samples, so it is not something that was in the samples (phenolics or excessive RNA, etc.).  I'm not sure what it is or how to get a clean pellet.  Any thoughts?

[phage434]

Did you do a chloroform extraction after the phenol/chloroform extract?  If I recall, the normal extraction following CTAB is chloroform only.

[dkkorzow]

phage434, on 22 March 2012 - 05:12 PM, said:

Did you do a chloroform extraction after the phenol/chloroform extract?  If I recall, the normal extraction following CTAB is chloroform only.

I did two different initial parts of the extraction - one was a phenol/chloroform followed by a chloroform and the other was just a chloroform extraction.  Both had CTAB and SDS in the solution.

[Aura]

Hi there. Have you solved the bubble problem? I'm asking because I'm getting this problem now. I have been doing the extraction for over 2 years without problems, so I was really upset thinking I was doing things wrong now. I found this thread [http://www.protocol-...osts/11355.html] where they suggest Trizol is contaminated. I am not using trizol but a home-made lysis buffer. It is not contaminated since my buffer does not form the bubble when I mix it with isopropanol. So, I'm guessing it is the phenol. I am changing my phenol bottle so I expect a cleaner extraction. Please, if you solved this, let me know.

-Aura-

Edited by Aura, 23 October 2012 - 12:04 PM.