10x Laemmli buffer - (Oct/12/2013 )
Is there any recipe for home-made 10x Laemmli buffer?
My sample is just too low concentration but the common stock is 2x,4x,5x and 6x...
And is it possible not to use powder form of SDS? I don't know how to dissolve it and we have 20% SDS in solution.
10X Laemmli buffer is impossible to make, since it would have to contain 100% glycerol, 625mM Tris-HCl (pH 6.8), 20% SDS (w/v), and 0.1% bromophenol blue. That is, it all adds up to more than 100%!
Not handling SDS in powder form is a good idea, because it's not good for your lungs. It would dissolve in the sample buffer though if you just mix with a stir bar for enough time. Just don't let it get cold, because the SDS will precipitate.
Thanks alot for the advice and i really agree with you
But i just found this, to make 10X stock here!
U think it will work?
To make 10 ml of 10x stock
- In 70 % glycerol / 30 % water, dissolve the following:
- 0.606 g Tris-base
- 2.5 g SDS
- Adjust the pH using pH indicator strips to 6.8
- Add 2 mg of Bromophenol Blue and make sure the powder is completely dissolved
- Adjust the final volume to 10 ml with 70 % glycerol / 30 % water before storing at -20°C.
- Add the appropriate volume of a β-mercaptoethanol 100% stock to your samples just before denaturing them at 95°C.
Sounds reasonable and worth a try. What volume are you going to load into each well? You can also make a thicker gel with a bigger comb. In my PhD lab we had big gel set-ups with wells that could take 50-80ul of sample.
I hope at least can fix 20ul...
Hopefully my lab has the big gel setting...:D
Have a nice day ~
sorry for the late reply ~