Isopropanol precipitation problem - (May/22/2009 )
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
you should not be seeing any two phase after addition of IP. maybe you have touched the bottom phase during phenol:chloroform step. Becareful while removing the top phase into another tube.
do you see the DNA pellet after centrifugation?
Go in with a pipette tip and draw the bubble off the bottom of the tube(s). Put it (them) in another tube and smell it -- is it phenol or chloroform?
Are you adding the RNAse before the chloroform and then the IP to the top phase?
Curtis: No pellets were seen after adding IP to the upper phase and centrifugation.
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
If your final step is an isopropanol precipitation, does the trace of phenol matter?
After adding the IP, mixing, and centrifuging at 13k rpm for 5 min, a DNA pellet should be present. Instead I have this bubble at the bottom. Could it be possible that a longer centrifugation is needed after the phenol:chloroform step? The protocol calls for a spin at 13K rpm for 3 min.
With phenol it is sometimes possible to have an layer inversion if the aqueous phase is dense due to very high salt concentrations. You think you are taking the aqueous layer, but it is on the bottom rather than on the top. This never happens with phenol/chloroform, where the chloroform density is quite high. Might this have happened to your samples?
High salt concentration in the sample...it could be possible. Before the phenol:chloroform step, 94 ul 5M potassium acetate are added to the mixture of ground tissue, extraction buffer and SDS, kept on ice for 5 min and then spin at 13k rpm for 5 min. The supernatant is then transfered to a clean tube and the phenol:chloroform mix is added. What is the purpose of adding potassium acetate? In some protocols I have used 7.5 M ammonium acetate.
I thought "bubbling" happens frequently after addition of isopropanol (IP) to the lysate? I observed the same thing with EtOH.
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.