I am facing some problem regarding the residual phenol contamination in my DNA samples. I use phenol-chloroform-isoamyl alcohol mix to remove proteins. The 260/280 ratio suggests the presence of phenol. What should be done to successfully remove the contaminants?
Avoiding phenol contamination depends upon your pipette handling. Careless pipetting may lead to minute contamination, but in your case if the values are seen in 260/280 ratio i guess it must be protein contamination and not phenol. Did you check for protein contamination?? Can you tell the values of your 260/280 ratio?
Repeat the procedure starting on adding chloroform alone. Be sure to mix the sample with chloroform so that any trace of phenol dissolves on the chloroform. Be very careful while transfering supernatant to new tube. Chloroform contamination is relatively easy to spot as small droplets precipitating to tube's bottom.
Hope it helps
According to my experience can traces of phenol be removed by use of disposable desalting columns filled with Sephadex G-25 /G-50 resin.
Be sure to use high quality polypropylene or the phenol will become "one with the tube". Good luck!
Dear Michael,
Firstly, thanks for your answer. Can you please elaborate on how phenol can become "one with the tube"? I think this is what has happened to me. Because, I have used the same protocol, but never have I faced such contamination. May be the tube was not okay and hence removal of phenol is being difficult.
Low quality polypropylene and old polypropylene can "bind" phenol. Also, low quality phenol can polymerize or contain phenolic acids. Please ask a chemist for the complete facts. Cheers!
In my experiments DNA isolation I do not use phenol - chloroform - isoamyl alcohol mix to remove proteins . I only use the chloroformo - isoamyl alcohol (24 : 1) and get very good results.
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