Temperature for EtOH Precipitation? - (Aug/10/2006 )
I have done Etoh Precipitaions in many labs and I have noticed there seem to be a few differnt ways of doing this. In the lab I work in now it is insisted that all reagents be cold and the spins are done at 4C. What is the reasoning behind this? I have heard that it is actually better to precipitate at room temperature. Also, does the sodium acetate HAVE to be at pH5.2? If it is at a higher pH will it affect the DNA yield?
doing allstuff at RT is enough, but cold enhance the DNA precipitation. i don't know what lies under that fact....
The pH of Na acetate is the important point. At that pH, DNA is more charged and can form easier some clumps that are important for DNA precipitation.
If pH is changed, the effect of na will decrease strongly.
Many say to precipitate in 4C. But me and my colleague used to do it at RT and everything was RT. It always worked.
May b for very low DNA conc. u might want to do it at 4C.
For low concentration DNA it's even better to put it at -80°C for 30 minutes . Goes faster.
Cold EtOH precipitation is old school thinking.
Tests done in the last 10 years have shown that RT precipitation yields the same results and that it's a lab myth that cold precipitation aids the precipitation or makes it precipitate faster or has a higher % yield.
If you're old school, like me, you are still comfotable with the cold precititation and eschew the new fangled RT precipitation. That's why some labs have the differences in temp of precipitation - it's a good gauge of which camp the lab manager is in.
You'll have to pry my cold EtOH out of my (very) cold, dead hands.
Rule of thumb:
If there are several different protocols for the same task (like precipitating DNA), none of them is critical. If most/all of them have a similar step (like buffer pH), it IS critical.
I have the feeling that precipitating in the cold gives a better yield.
I say the feeling, because I never compared two tubes from the same batch.
However, once the DNA is precipitated, you can wash with RT ethanol.
this is usually the case when you use kits. you precipitate with isopropanol, and wash with ethanol (at room temperature)