concentration of magnesium in the biomix red protocol

[Silvia_84]

Hi all,

 

I am doing PCR and I am using the biomix red as a polymerase. The mix already contains all the components for the reaction but I need to increase the concentration of magnesium in my reaction. I can't understand very well the protocol. It says that the MgCl2 concentration in the buffer provided is 6mM (3mM final concentration) and should be adjusted just if necessary. Then it reports a table showing the volume of additional MgCl2 to add to a 50 ul reaction to achieve different final concentrations. The table says to add 0 ul of 50 mM MgCl2 to 50 ul final reaction volume to reach a concentration of 2mM, 0.5 to reach a concentration of 2.5 and 1 for a 3mM concentration. What I don't understand is that reading this protocol it seems to me that the reaction mix already contains 3mM of Magnesium. So why should I add 1 ul to reach a final concentration of 3mM?And if I wanted a 5mM concentration, should I add 3ul? I was not able to attach the link, anyway if you google biomix red protocol , you find it right now.

 

I hope to have been clear.

 

Thank you

[foxy john]

Not sure but just try it and see. Strongly suspect that as long as you have Mg ions present it's OK.