resolution of similar size products using agarose gel - heeeeeelp !!! =P (Oct/20/2005 )

Hello!

I need to separate products with similar sizes from the same PCR reaction. The sizes are 2200 and 2150 (aprox) and 1800 and 1850 (aprox). To make things a little bit worse (yay!) I also have a 600 (aprox) product in the tube that I can`t lose...
Am I doomed, or can anyone here give me a few tips to save my thesis???

Thanx ....

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hi
you have to cast a big gel (best is resolving on 15cm), using 2% agarose

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Indeed, a big gel (I'd say the bigger the better), about 2% agarose and run it at a low voltage.

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You can gel purify to get your 600 bp band. The 1800-1850 and 2200- 2150 bands will not separate efficiently on a 1% agarose gel. Do you have unique restriction sites incorporated on either of them? If not, you could always go ahead with inserting into your plasmid, transforming, and just sequence 10 or so preps. You should get a couple of the correct isolates.

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You should also check out:

1. Brody, J. R. and S. E. Kern. 2004. Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. BioTechniques 36:214-216
2. Brody, J. R., E. S. Calhoun, E. Gallmeier, T. D. Creavalle, and S. E. Kern. 2004. Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media. BioTechniques 37:598-602.

In addition to being a faster medium, gels of this type are superior at resolving small size differences (see especially reference 2).

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Go for a big gel... you can also try some agarose that allows 4% gels. Should increase the resolution.

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50 basepairs is not that small difference for a 2 kb band. Just run the gel long enough.
Have a µl EtBr in your DNA before loading, and no EtBr in gel and buffer (increases the contrast between band and gel), run the gel quite slowly for as long as you can before DNA runs out of the gel. check your gel regularly (but do not keep it on UV for long times if you want to clone the products).

Any chance of just cloning the entire PCR collection and let the bacteria do the sorting?
It should be possible to get different clones with only one of the inserts in each.
This have been my method of choice if the PCR products are to be cloned anyway.

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thanx for the tips. actually i dont need to purify these products (yet!), i just have to be able to be 100% certain that i have one band or the other in lots and lots of samples, that's why i need to resolve them well, so theres no doubt about which product im seeing.
here we dont use br-et (forbidden by the "im scared of cancer" doctorate students here), we use cyber green en the gel, is that a problem?
well, thanx to all, cya round and tell ya if theings worked or not (if not, ill ask for more tips)

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hi
it's not a problem to use cyber green. It's actually supposed to be more precise than EtBr and surely less toxic.

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Hi there,
Do you prepare the Lithium buffers by yourself? Do you mind posting your recipe (buffers for dummies style...) I ask because I have good results with the Sodium boric acid buffer but I can not get good resolution with the Lithium buffers, maybe I am doing it wrong.
Thank you
Clarice

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