adding too much phenol chloroform? - (Jun/16/2007 )

hello,
If I am trying to isolate my DNA insert from a plasmid vector, and I put in twice as much phenol/chloroform/isoamyl alcohol (25:24:1) than the required amount, what will this generally do to my DNA or plasmid vector? basically, what does too much phenol/chloroform/isoamyl alcohol do to your DNA or vector?

to DNA, nothing.

However the cost goes up and the boss may become unhappy.

haha.... yea.. no harm to your DNA. But I am sure the amount used is quite little too right?

The phenol is for separate the nucleic acids from the proteins, so it will not do anything and you will not get more DNA (just cleaner). If you want to get more quantity of DNA just add another vol ume of lysis buffer after you get the aquous layer, mix and centrifuge.

sometimes i use phoH:chcl3 twice of the vol of sample soln when i see too much protein in my preparation. i think twice volume work more efficiently than equal vol. in this case. am i right???

If you have lots of protein then is better to use 2 vol.