solution preparation - (Jul/19/2016 )

Hi,

I'm trying out a site directed mutagenesis kit and I have to prepare the following:

100mM KCl

100mM (NH₄)₂SO4

200mM Tris-HCL (pH8.8)

20mM MgSO₄

_{1% Triton X-100}

_{1mg/ml nuclease free BSA}

_{I understand that if I prepare 10ml of the above stock of buffer, I would need the following amounts but how much Tris-HCl do I add. Do I first I prepare a solution of 200mM Tri-HCL to pH 8.8, but then how much volume do I add to the 10ml stock that I'm making. Or do I just add 315mg of Tris-HCl powder (200mM in conc)?}

100mM KCl= 74.5mg

100mM (NH₄)₂SO4 = 132mg

200mM Tris-HCL (pH8.8)=??

20mM MgSO_4=24mg

_{1% Triton X-100= 100ul}

_{1mg/ml nuclease free BSA= 10mg}

you're better off preparing a 1M stock of tris-hcl, pH 8.8 and adding 2 ml to the rest and adjust to 10 ml.

there are a couple of ways to prepare the tris-hcl stock:

mix 1M tris base and 1M tris-hcl until pH 8.8 is attained

or

weigh out tris base for 1M for a specific final volume, suspend to less than the final volume, adjust the pH with hcl (get close with concentrated then use diluted hcl to reach the final pH), then adjust to final volume.