Isopropanol precipitation problem
Posted 22 May 2009 - 01:30 PM
We started using a protocol for plant DNA extraction in which there is a step for precipitating the DNA from the samples using isopropanol. The problem is that after briefly mixing the isopropanol with the aqueous DNA suspension, a bubble forms at the bottom of the microtubes. It looks like if there are two phases, organic and inorganic.
Briefly, the protocol uses:
An extraction buffer (Tris, NaCl, EDTA), SDS, heating samples at 65 C, adding 5M potassium acetate, extraction with phenol:chloroform, chloroform, RNase, and then isopropanol. Spining time during the organic steps is for 3 min at max rpm. What could be forming the bubble, some phenol residue?
Any help is appreciated
Posted 23 May 2009 - 08:25 AM
do you see the DNA pellet after centrifugation?
Posted 23 May 2009 - 10:22 AM
Posted 24 May 2009 - 09:58 PM
Edited by juanboja, 24 May 2009 - 09:58 PM.
Posted 25 May 2009 - 09:25 AM
I did as HomeBrew said, and the bottom bubble smells like phenol!!
Juanboja: I transfered the upper phase to a clean tube after the chloroform step, and then added RNase.
It appears that I am taking phenol after the phenol:chloroform step!! Well, I have done this hundreds of times using other protocols without problems....It seems that I'm getting old
Thanks a lot guys
Posted 25 May 2009 - 11:19 AM
Posted 25 May 2009 - 11:35 AM
Posted 26 May 2009 - 04:41 AM
Posted 26 May 2009 - 07:21 AM
Posted 29 May 2009 - 07:24 AM
In genomic DNA precipitation, addition of alcohol will frequently result in "aggregation" of DNA to form thread that you can see with naked eye after inverting the tube to mix. I see this most of the time.
I'm assuming that the potassium acetate (or high salt) used here is for salting out proteins and the organic solvent is to enhance the sequestration of protein from the aqueous phase.
Just curious, you're extracting DNA from plant material? Is it from fruits or leaves? I used to extract DNAs from both fruits and leaves and it's a bummer to avoid polyphenol (or phenolic compound) contamination as well as its oxidation that will irreversibly bind to DNA and causing unretrievable or unusable DNA for downstream application like PCR etc. Heating, it seemed, exacerbates this oxidation. I remember that I dreaded using reducing agents such as beta-mercaptoethanol to avoid such oxidation. However, some papers used PEG, CTAB method etc....
Hope this input will be of some help.
Edited by lsek, 29 May 2009 - 07:25 AM.
Posted 10 June 2009 - 11:08 AM
Thanks for the input...
Tubes on the left show the "bubble" (tomato), samples on the right without "bubble" (cucumber seedlings)