Phenol-Chloroform Extract vs Commercial Kits - (Jun/20/2008 )
Dear all,
I've never used the phenol-chloroform method to isolate and purify my DNA. Now, using the commercial products (Promega) seem easy but it isn't perfect - based on my sequencing results. Read that the former method give a better DNA yield (cleaner), are there any other drawbacks beside phenol being hazardous?
Using these commercial kits made me feel like a robot programmed to do DNA purification from bacteria as I don't know the constitutions of some reagents. Do they use phenol at all?
I've also learned that the A260:280 / 260:230 ratio gives indication of the purity of DNA, so how do I go about washing the DNA which is already in solution?
Thank you.
Hi Dreamchaser,
I sometimes used the Phenol Chloroform extraction for doing miniprep, especially when I need a high yield. The disadvantage of this method is in my opinion not only the toxicity of Phenol/Chloroform but also that leftovers may cause troubles with enzymes (PCR digestion etc). After the Phenol/Chloroform precipitation I always add a Ethanol-precipitation tho remove phe/chl residues. But even then I sometimes had troubles with my digestions. For sequencing our sequencing service strongly recommends that we use kits. For kit preparation no phenol or chloroform is needed because the principle of these kits is, that DNA can bind to the column material in pH/salt dependent manner.
Another point for kits is the big time saving, the preparation is much faster. And they became a lot cheaper in the last years (this was alsways the point of my boss: kits are too expensive, so use the Ph/Chl-method).
no, if you are into the "transfer the upper phase without disturbing the lower" and know how long it takes the Ethanol to evaporate!
I prefer the "classical" method, as you can modify it for each application. If you want to take the time "playing" around with optimum incubation time/centrifugation time/concentration for your specific sample your methods usually get better and even faster! I usually avoid using phenol and use chloroform/isoamylalcohol (not that toxic) with good results.
a friend of mine lost one year of her PhD due to a kit where they changed the manufacturer of spin columns...worked fine for two years and suddenly - nothing worked anymore! So she tried, got replacement, tried again, and suddenly a few month were over!
But usually these kits are said to be without toxic stuff....but there seems to be a recommendation to wear protective clothing in all of them
But when it comes to the "to use kits or not to use them" question I am a little bit biased
(as usual 
)You want to get rid of proteins?
I've never used the phenol-chloroform method to isolate and purify my DNA. Now, using the commercial products (Promega) seem easy but it isn't perfect - based on my sequencing results. Read that the former method give a better DNA yield (cleaner), are there any other drawbacks beside phenol being hazardous?
Using these commercial kits made me feel like a robot programmed to do DNA purification from bacteria as I don't know the constitutions of some reagents. Do they use phenol at all?
I've also learned that the A260:280 / 260:230 ratio gives indication of the purity of DNA, so how do I go about washing the DNA which is already in solution?
Thank you.
its better to use a kit when you r going for sequencing. You can repeat your washing step during plasmid prep to remove any more contaminants left.
cheers
Well, spent my undergrad Finals time doing purification using kits (thank God for my supervisor!) but then it'd be nice if the phenol-chloro method was used. Will definitely try the "classical" method employing isoamyl alcohol.
Though its much faster and easier using the kits, it doesn't always guarantee a pure DNA. So, yes, I'm trying to get rid of
protein if A260:280 isn't in the optimum range. If the DNA is still adsorbed to the silica in the spin column, I could add
ethanol and spin them (Wash step). What do I do if my DNA has been eluted into TE ready for the next steps (sequencing etc.)?
Thanks people, for the explanations.
Though its much faster and easier using the kits, it doesn't always guarantee a pure DNA. So, yes, I'm trying to get rid of
protein if A260:280 isn't in the optimum range. If the DNA is still adsorbed to the silica in the spin column, I could add
ethanol and spin them (Wash step). What do I do if my DNA has been eluted into TE ready for the next steps (sequencing etc.)?
Thanks people, for the explanations.
Ok guys. What would you say if I offered you a solution that carries the precision and efficiency of classical phenol/chloroform method together with the speed and convenience of commercially available extraction kits? Using it still lets you be the boss because there is plenty of room for adjusting the protocol...
Excited to hear what you are going to offer...
Dear all,
We developed a Smart Helix protocol for fast and effective nucleic acid extraction, using physical and chemical cell beating/disintegration method coupled with phenol/chloroform extraction. The protocol is far more effective in extraction compared to competitor kits and it allows quantitative analyses of airborne bacteria, direct qPCR analyses of bacteria in potable water, detection of avian influenza virus in soil samples etc. Whenever extraction efficiency due to low target NA content is in question, Smart Helix can be used for a reliable and quantitative rasult.
The protocol is in the stage of commercialization at the moment. It is available in sample kits for the curious ones. Anyone interested in testing the new protocol is invited to contact us through the company email venturia@venturia.si. We will gladly provide some reference data collected through testing the protocol and discuss the topic with you.
Best regards,
Venturia