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10x Laemmli buffer


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#1 wdyeo

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Posted 12 October 2013 - 09:27 AM

Dear all, 

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.

Thank you.



#2 doxorubicin

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Posted 12 October 2013 - 01:15 PM

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. 



#3 wdyeo

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Posted 14 October 2013 - 02:57 AM

Hi doxoburicin, 

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?

 

http://openwetware.o...is_formulation)

 

Variant

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.

 

 

Thank you.



#4 doxorubicin

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Posted 14 October 2013 - 03:51 AM

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.



#5 wdyeo

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Posted 21 October 2013 - 04:35 AM

Hi, 

Yeah...maybe...

I hope at least can fix 20ul...

Hopefully my lab has the big gel setting...:D
Have a nice day ~



#6 wdyeo

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Posted 21 October 2013 - 04:36 AM

sorry for the late reply ~






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