13 replies to this topic

[Mad researcher]

What's the difference between using TAE or TBE buffer for electrophoresis?

And what is the difference between using 1X TAE and 5X TAE Vs 1X TBE and 5X TBE buffer?

[leelee]

I use my TAE buffer (1X) for normal, shorter electrophoresis runs (PCR products, plasmid digests etc) and then TBE at 0.5X for RFLPs that I need to run overnight or longer (doesn't get as hot as the 1x TAE).

Never really questioned why, that's just how its done in my lab.

The 5X solutions will be stock solutions, that you need to dilute prior to using.

Edited by leelee, 10 September 2012 - 11:53 PM.

[Mad researcher]

don't you use TBE buffer for running electrophoresis?

[bob1]

TAE is tris-acetate-EDTA, TBE is tris-borate-EDTA, they and many other buffers can be used for electrophoresis.

[BioMiha]

As leelee said TBE causes less heating because borate ions have better mobility than acetate. The problem with using borate is if you want to excise your band out of the gel and purify it the borate ions form complexes with the DNA and the yield is much lower. Therefore people use TAE. There was a paper on this subject a while ago called "Not your father's buffer" and it said that the best is Li-borate or Na-borate and in my experience you can really speed things up with a different buffer.

[Mad researcher]

Thanks a lot guys.
@Biomiha - Not your father's buffer - nice article. May be i will try using Na-Borate buffer instead of TBE and TAE

[ascacioc]

I am totally for the SB buffer (better resolution) Tris based buffers are so passe

To add to the things above: you can use both for agarose electrophoresis; but when I did acrylamide electrophoresis of short DNA fragments, I used TBE. More, in the case of urea denaturating gels, when the urea is not enough to obtain ssDNA, some people recommend to run everything (and prepare the gel as well) in 2X TBE to increase heating hence melting.

[Trof]

There was this routine to use 1x TAE gels for fragments cut out and 0.5x TBE for all other elfo, but I just smashed this tradition, because I've seen no difference in yields between the buffers (using Qiagen MinElute kit) and TAE is a worse buffer. Also not completely uninportant thing is, TAEs had to be prepared for each run from 10x solution, unlike the 0.5 TBE that we have in big tank ready to use

I'm getting more curious about the SB buffer, you all mentioned beter resolutions, possible higher running voltages and stuff, but I'm also concened about the isolation from such gels, yields, salt content (our main aim is for sequencing) stability of DNA and so on. And generaly, does it even have any disadvantages at all?

[bob1]

@Trof - I use SB for gel extraction and subsequent cloning and have had good yields with the Axygen gel extraction kit.  I tended to get poorer yields when using TBE, but that may have been due to loading lower amounts on the gel or using a different extraction kit.

The DNA seems to be pretty stable, but I havn't assessed true integrity by mass spec or looking for crosslinking or anything else.

[Trof]

So far I searched the forums and made this list of SB buffer features:

pros:
- lower conductivity, less heating, higher voltage/speed possible
- better resolution (under 3 Kb)
- price (as I understand)

cons:
- lower buffering capacity, easily depleted, reuse not possible
- problems in high sample salt concentrations (possible problems in running restriction fragments?)
- not suitable over 3 kB

not sure:
gel extraction yield (particulary interested in comparison with 0.5x TBE and Qiagen kits)

Also I found more versions of protocols.
boric acid + NaOH pH 8.2
boric acid + NaOH pH 8.5
sodium tetraborate + boric acid

Which one is better?

I'm kind of decided to do extensive testing (sharpness, resolution including very small bands, reusability, gel extraction yield) it would be better for a student but I don't have one now, but there is a new colleague that seems to be yet unoccupied so I ask if she's interested.
Our primary goal will be testing the gel extraction yields and purity, because otherwise I'm not aware about any serious resolution issues with your TBE (well didn't see anything from SB yet, maybe I'll change my mind) apart from <100 bp fragments of course, and running on higher voltage isn't really that important for us.

[bob1]

I reuse my SB several times before changing, probably just as frequently as I would have changed my TBE.

[Mad researcher]

i am still using TBE but i don't reuse the TBE more than 3-4 times.
i will switch to SB buffer and try that as well.

[Trof]

I just got to situation when I need to distinguish on gel restriction fragments of very small PCR product (39 + 54 bp from uncut). As I know how problematic is TBE gel in fragments with length < 100 bp, I was thinking that trying SB buffer for this would be handy.

But problem is, it's a restriction reaction, so high salt content. These have pretty problems even in TBE gels, mostly smaller than 200 bp are just smears, because when the gel runs long enough to clean from salts, the smaller fragments are just too blurry.

I was thinking maybe desalting the samples before loading would help, but I can't use common Qiagen columns for that as their lower limit is 70 bp. Precipitation is probably a way to go, but the fragments are small and the concentration wouldn't be high either.

[phage434]

Try 2-3% Nusieve 3:1 agarose or Metaphor agarose for high resolution, or a Tris PAGE gel.  You could desalt with drop dialysis, which is fast and easy.