Hello, my name is Chris Lemke.
I work for the life science business of Merck KGaA, Darmstadt, Germany.
I'm a Research Technology Specialist and
I live in Charlotte, USA.
In this video, I will discuss gel electrophoresis buffers,
specifically, why are they needed?
What are the most common types?
And which type is right for your application?
So when you're running a gel,
the buffer is needed for two primary reasons.
First, the buffer provides ions
and these ions carry the electrical current
through the gel.
But second, the buffer acts as a buffer,
maintaining a fairly constant pH throughout the gel run.
Now the two most common buffers used
for nucleic acids are Tris Acetate EDTA
and Tris Borate EDTA.
So what's the difference between these two buffers,
and which of these buffers is right
for your application?
TAE Buffer is a solution consisting of Tris Base, acetic acid, and EDTA.
TAE Buffer is commonly prepared as a 50X concentrated stock.
To make the stock, dissolve 242 grams of Tris Base into distilled deionized water.
Add 57.1 milliliters of Glacial Acetic Acid.
Then add 100 milliters of 0.5 molar EDTA solution at a pH of 8.0.
Then bring the final volume up to 1 liter total, using distilled deionized water.
TBE Buffer is a solution consisting of Tris Base, boric acid, and EDTA.
TBE Buffer is commonly prepared as a 10X concentrated stock.
To make the stock solution, dissolve 108 grams of Tris Base and 55 grams of boric acid
into 900 milliliters of distilled deionized water.
Then add 40 milliliters of 0.5 molars EDTA solution at a pH of 8.0.
Then bring the final volume up to 1 liter total using distilled deionized water.
So which buffer is right for your application?
TBE buffer has better buffering capacity than TAE,
so if you'll be doing extended long runs
or repeated runs in the same buffer,
you'll want to use TBE buffer.
TAE buffer has better conductivity than TBE,
so DNA fragments will migrate faster in TAE buffer than TBE.
TBE buffer supports better agarose cross-linkage,
so you'll get better resolution of large DNA fragments in TBE buffer
and better resolution of smaller DNA fragments in TAE buffer.
The borate in TBE buffer inhibits many common enzymes used in molecular biology,
so if you'll be using the DNA for any downstream applications involving an enzyme,
like PCR or clonal ligation,
you'll want to use TAE buffer.
And finally, TBE buffer costs more to make than TAE buffer.
Thank you for watching.
I hope this helps.