DNA ligation - problem with purification (Feb/14/2007 )
I'm performing DNA ligation after a step of phosphorylation.
After the phosphorylation of my DNA I purify it with a column and the DO and quantity are ok. So I perform my ligation with the T4 DNA ligase according to the manufacturer prescriptions. Next, I extract the DNA from the reaction mixture with Phenol and I precipitate it wiht Ethanol and Sodium acetate. Finally I suspend DNA in 10µl sterile water. When I perform a measure of the DO, I observe very great amount of DNA (5µg/µl) but the ligation was made less thant 1µg....so ok i think that I have a contamination...maybe... but where?? I measure the DO of all my reagents and here is my problem: the ligation buffer has the same DO that DNA 260/280nm =2 and the nanodrop calculate an amount of 5µg/µl DNA in the buffer is-it possible??? it's a very great contamination I think...
Please help me.
The ligation buffer contains ATP. Ethanol precipitation is not removing all of it. This results in higher than expected OD values when you measure the OD of the ligation reaction.
after ligation, you don't need to clean up the DNA.Actually is best if you don't, as there is a very real risk of loosing your DNA.
Just ethanol percipitate the DNA, wash off the salt with 70% Ethanol, resuspend in sterile distilled water and transform.
Way too much effort (and danger of creating problems). Directly transform the ligation reaction. If you need to run it on a gel, heat inactivate at 80C for 20 minutes. The only reason to purify the ligation reaction DNA is to electroporate, but even then drop dialysis works with much less effort than these techniques.
One doesnt need to use too much DNA for ligation. I would normally use about 20-40 ngs, total DNA conc.