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whusean

SDS PAGE HELP!!!!!

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Hi people<br style="font-family: verdana, geneva, lucida, 'lucida grande', arial, helvetica, sans-serif; font-size: 13px; background-color: rgb(245, 245, 255); "><br style="font-family: verdana, geneva, lucida, 'lucida grande', arial, helvetica, sans-serif; font-size: 13px; background-color: rgb(245, 245, 255); ">Im a 3rd year biomedical student currently writing my dissertation. I am confused about how to correctly write the composition of my stacking/separating gel. My supervisor told me all of the components e.g. Tris must be expressed as concentrations and not volumes, but im having trouble working this out.<br style="font-family: verdana, geneva, lucida, 'lucida grande', arial, helvetica, sans-serif; font-size: 13px; background-color: rgb(245, 245, 255); "><br style="font-family: verdana, geneva, lucida, 'lucida grande', arial, helvetica, sans-serif; font-size: 13px; background-color: rgb(245, 245, 255); ">The composition of my stacking gel was:<br style="font-family: verdana, geneva, lucida, 'lucida grande', arial, helvetica, sans-serif; font-size: 13px; background-color: rgb(245, 245, 255); "><br style="font-family: verdana, geneva, lucida, 'lucida grande', arial, helvetica, sans-serif; font-size: 13px; background-color: rgb(245, 245, 255); ">7 ml 30% Acrylamide / 0.8% Bisacrylamide<br style="font-family: verdana, geneva, lucida, 'lucida grande', arial, helvetica, sans-serif; font-size: 13px; background-color: rgb(245, 245, 255); ">12.5 ml 4X Tris<br style="font-family: verdana, geneva, lucida, 'lucida grande', arial, helvetica, sans-serif; font-size: 13px; background-color: rgb(245, 245, 255); ">25.5 ml H20<br style="font-family: verdana, geneva, lucida, 'lucida grande', arial, helvetica, sans-serif; font-size: 13px; background-color: rgb(245, 245, 255); ">10 ul 2% bromophenol<br style="font-family: verdana, geneva, lucida, 'lucida grande', arial, helvetica, sans-serif; font-size: 13px; background-color: rgb(245, 245, 255); "><br style="font-family: verdana, geneva, lucida, 'lucida grande', arial, helvetica, sans-serif; font-size: 13px; background-color: rgb(245, 245, 255); ">Could anyone help me understand the correct way i should express this in my final report. <br style="font-family: verdana, geneva, lucida, 'lucida grande', arial, helvetica, sans-serif; font-size: 13px; background-color: rgb(245, 245, 255); "><br style="font-family: verdana, geneva, lucida, 'lucida grande', arial, helvetica, sans-serif; font-size: 13px; background-color: rgb(245, 245, 255); ">Thanks

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Sorry about that i dont know what happend there. Basically what I said was;

 

Im currently writing my final year dissertation and I need to write up about an SDS page i did. My supervisor told me to write the composition of stacking/seperating gels as concentrations and not volumes. Im having trouble calculating these concentrations, so a bit of help would be much appreciated. I just need to know how to properly write it in my final project. The composition of my stacking gel was:

 

7ml 30% acrylamide/ 0.8% bisacrylamide

 

12.5 ml 4X Tris buffer

 

25.5 ml H2O

 

10 ul 2% bromophenol

 

Thanks in advance

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I am not sure how much I can help. However, the concentration of acrylamide and bisacylamide are given as a ratio of mass to volume. For example, if I had 10% acrylamide then it means that 10 grams of acrylamide have been dissolved in 100ml of a solvent. If I have 8 ml of this acrylamide, can you now guess how much it contains? And if I now add suitable buffers to a new volume, I can then recalulate how much acrylamide and bisacrylamide there is in the final solution as grams per 100 ml of the new solution. I hope this makes sense.

Edited by jimmydasaint

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I'm not following you to be honest...is there any chance of you explaining it again, in a slightly different way. Many thanks

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I hope I am Ok in doing these calculations so I will prepare an exemplar. Others can correct me if my method needs correction. Mods, if I am too detailed in my advice, then I apologise in advance. Let's imagine that you have:

 

12 ml 20% acrylamide/ 0.8% bisacrylamide

 

12.5 ml 4X Tris buffer

 

25.5 ml H2O

 

10 ul 2% bromophenol

 

Then, 20% acrylamide + 0.8 bisacrylamide = approximately 20.8 g/100 ml solvent

 

= 0.208 g of acrylamide:bisacrylamide per ml.

 

I have 12 ml, therefore I have 12 x 0.208 g total amount of acrylamide/bisacrylamide.

 

Then this amount is spread over 12 + 12.5 + 25.5 ml in total. Therefore you can now convert the total number of grams acrylamide:bisacrylamide in 100 ml of solvent. (bromophenol blue is negligible in these calculations). Does that make sense?

Edited by jimmydasaint

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I think i understand it now..thanks alot. Just one more quick question....once i get the conc in mg/ml, it would then need to be converted into mg/100 ml in order to get a % conc value right?

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Yes. In my example,only slightly related to yours,

 

12 ml 20% acrylamide/ 0.8% bisacrylamide

 

12.5 ml 4X Tris buffer

 

25.5 ml H2O

 

10 ul 2% bromophenol

 

Then, 20% acrylamide + 0.8 bisacrylamide = approximately 20.8 g/100 ml solvent

 

= 0.208 g of acrylamide:bisacrylamide per ml.

 

I have 12 ml, therefore I have 12 x 0.208 g total amount of acrylamide/bisacrylamide = 2.496 g

 

Then this amount is spread over 12 + 12.5 + 25.5 ml in total = 50 ml

 

So there are 2.496 g per 50 ml and therefore 4.992 g/ 100 ml, making it an approximately 5% SDS/PAGE gel which I assume you need to separate out high molecular weight macromolecules? If not, I have messed something up in the calculations.

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As an additional note, many dislike the "x"-buffer notation, as sometimes errors are in the actual recipe. It is preferable to a) indicate the molarity and b) the pH. Only exceptions are pre-made buffers, in which case the manufacturer should be stated.

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