I did something stupid - 10 mM dNTPs vs 2.5 mM dNTPs (Sep/29/2008 )
Hi All,
So, I ran out of a Green Master Mix that had been spoon-feeding me adequate results. However, in an attempt to be thrifty, I rummaged through our stores and found the individual components that make it up (5x Green (or Colorless) Rxn Buffer, dNTPs, Taq, etc.) Well, thinking I'd make a whole to batch from scratch, I used all of the buffer and with it, instead of using 2.5 mM dNTP mix as my 'recipe' called for, I used 10 mM dNTP mix. I didn't figure this out until I ran a gel and got NOTHING. NADA. (Except for some beautiful ladders) I imagine my dNTPs are chelating all of the Mg++ (what do you think?) and I was wondering if anyone knew of any way I could rescue this concoction, since, at the moment, I have no additional Rxn Buffer (other than what is in my failed batch)? Could I theoretically add enough MgCl2 to make the system work again? Or will I run into an upper limit before that?
This stinks.
Thanks to everyone. Cheers...
Synthasome
Find out what the reaction buffer is, make some at 1x, and add three parts of it for each part of your 4x mix. Add Taq and Mg to suit.
So, I ran out of a Green Master Mix that had been spoon-feeding me adequate results. However, in an attempt to be thrifty, I rummaged through our stores and found the individual components that make it up (5x Green (or Colorless) Rxn Buffer, dNTPs, Taq, etc.) Well, thinking I'd make a whole to batch from scratch, I used all of the buffer and with it, instead of using 2.5 mM dNTP mix as my 'recipe' called for, I used 10 mM dNTP mix. I didn't figure this out until I ran a gel and got NOTHING. NADA. (Except for some beautiful ladders) I imagine my dNTPs are chelating all of the Mg++ (what do you think?) and I was wondering if anyone knew of any way I could rescue this concoction, since, at the moment, I have no additional Rxn Buffer (other than what is in my failed batch)? Could I theoretically add enough MgCl2 to make the system work again? Or will I run into an upper limit before that?
This stinks.
Thanks to everyone. Cheers...
Synthasome
You are probably right, the dNTP is probably chelating all the MgCl2. Could you get / buy / make more Rxn buffer? You are going to do more PCRs right? So you might as well make a bigger stock of Master mix at the correct concentration.
You could increase the MgCl2 as you have suggested until the system starts working again. But that would take a few trials. I am unsure how the Taq will react at high concentrations of Mg2+ ions.