different percent gels - question (Oct/26/2008 )
Can someone explan to me how you possibly choose what percent gel to run your DNA, RNA or proteins at? In school, someone will just tell you to run a 0.8% PAGE to see your DNA' or a 10% SDS PAGE gel to see your proteins. However there are varying percentages of both these types of gels. Can someone inform me as to why a certain percent gel is better over another and what percent gel is used in a particular situation?
as for DNA we use TAE, TBE and other sort of buffer . PAGE is for differentiatting very very small difference in bp.
% of gel provide diff sepearation and resolution .
lower the % gel u can separate huge size DNA better but at the expense of having blurry band esp for small DNA due to diffusion .
higher % is good for separating small DNA with good resolution .
as for the % gel to use for DNA . i believe there's a guideline in the russell and sambrook(might spell wrongly) molecular cloning book.
i have done from 0.5% up to 2% for DNA in 1x TAe.
Normally for plasmids you use low percentage gels, so about 1% or lower, depending on the size of the plasmid. If you have small DNA fragments from pcr (say 200 bp) you use 2% gels.
For protein gels, it depends on the size of the protein you want to detect. Standard gels are 12,5 %, these give good solution for 60-80 kDA, if you have bigger proteins, you use 10% or lower (down to 8%, 6% for really big ones about 120-150 kDa). If you have small proteins about 20-50 kDa, 15% gels will be better.
think of it as gelatin (jelly desert). the more concentrated it is the harder the inside fruit will move, instead, if you use too little then it is most likely the fruit won't stay in place.
just to be a bit more graphical:
Here is a good web page for guidelines when choosing gel concentrations:
http://www.clontech.com/support/tools.asp?...;tool_id=155811
Look in the Lab FAQs from Roche:
https://www.roche-applied-science.com/techr...bfaqs/index.htm
Originally you were able to get a hard copy. Now it is only available online. However, on page 10 they tell you what percentage gel you need for which DNA fragments
Thanks all for the help.
One more question:
I just ran pUC DNA on a 2.5% agarose gel. On the gel, it looks like very high MW meaning it did not run very far into the gel. Does this mean that I should have used a lower concentration % gel?
or that you used a very high voltage, or didn't let it run long enough. when using 2% agarose gels i could easily detect bands of 600 bp if that is any help.
Yes, it was too big. Use 1% for plasmid as a standard for beginning.
You need to understand that the agarose or acrylamide in the gels are creating pores that the sample runs through. This is how the sample gets separated by size. The more you use (higher percentage) the smaller the pore size. This is great for separating smaller things (DNA or proteins) but at the expense of resolving the larger stuff. This is why you saw the larger DNA hardly move in the 2.5% gel. The less agarose or acrylamide used (low percentage) the larger the pores. This is great for resolving the larger stuff but the smaller stuff just runs right through and doesn't separate well. The percent of gel to use depends on what you are trying to resolve. For "normal" sizes (DNA: 1kb-8kb and protein: 20kDa-100kDa) I use a 1% agarose and 10% acrylamide. If I am specifically wanting to purify or examine anything bigger or smaller I will consider using a lower or higher gel respectively. The tough part is when you need the two extremes. There are precast gels you can buy or equipment to let you pour gels (just buy them!) that have a gradient to separate high and low when needed.