whusean Posted November 27, 2011 Share Posted November 27, 2011 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 Link to comment Share on other sites More sharing options...
Schrödinger's hat Posted November 27, 2011 Share Posted November 27, 2011 May I introduce you to the preview post button? I don't believe you have met. Link to comment Share on other sites More sharing options...
whusean Posted November 27, 2011 Author Share Posted November 27, 2011 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 Link to comment Share on other sites More sharing options...
jimmydasaint Posted November 27, 2011 Share Posted November 27, 2011 (edited) 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 November 27, 2011 by jimmydasaint Link to comment Share on other sites More sharing options...
whusean Posted November 27, 2011 Author Share Posted November 27, 2011 I'm not following you to be honest...is there any chance of you explaining it again, in a slightly different way. Many thanks Link to comment Share on other sites More sharing options...
jimmydasaint Posted November 27, 2011 Share Posted November 27, 2011 (edited) 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 November 27, 2011 by jimmydasaint Link to comment Share on other sites More sharing options...
whusean Posted November 27, 2011 Author Share Posted November 27, 2011 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? Link to comment Share on other sites More sharing options...
jimmydasaint Posted November 27, 2011 Share Posted November 27, 2011 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. Link to comment Share on other sites More sharing options...
whusean Posted November 27, 2011 Author Share Posted November 27, 2011 Yeah thats great, much appreciated Link to comment Share on other sites More sharing options...
CharonY Posted November 27, 2011 Share Posted November 27, 2011 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. Link to comment Share on other sites More sharing options...
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