Buffer for 5 Kb DNA fragments - (Aug/23/2009 )

Hi all,

We have been using Tris borate edta buffer for Agarose gel electrophoresis of plasmid DNA of size 3 - 5 Kb.

Recently i have read lithium buffers give best resolution. Which buffer shall i use for our DNA isolation, lithium borate / lithium acetate / sodium borate and at what conc. We use 1% as our gel conc. is that O.k. Kindly guide.

thanks in anticipation.

biotef on Aug 23 2009, 07:58 PM said:

The Maniatis suggest TAE buffer and it works great up to even 20KB size. Just a note TAE cannot run as fast as TBE because of por buffer capacities and heat is getting the gel to melt if run too fast. I recommend to run around 100 volts not more
TAE stand for Tris Acetate Edta buffer
http://www.thelabrat.com/protocols/16.shtml

biotef on Aug 24 2009, 01:58 PM said:

A couple of questions/points:
1. What are you trying to resolve? 5 kb vs 3? 5 kb vs 5.5 kb? I would have thought 1% might be a bit high, and you could go down to 0.8% even, unless your fragments are very close in size.; in that case, I'd say stay with 1% but run the gel out a long way.
You also need to remain aware of not overloading your lanes.

2. I haven't used lithium gels, so I can't comment on them. We have had some success using borate gels, because you can run much faster without heating up the gel to melting point (most of the heating is caused by the Tris). You should be aware, however, that borate really doesn't offer any significant buffering capacity. Our recipe for a 20x stock is 38 g/l sodium borate, 24 g/l boric acid. You also get nice high resolution, definitely at the low size range (say, under 1kb), but also up where you are.

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