If your 3600bp fragment has some restriction sites for particular enzymes, which aren't exist in another fragment, you would be able to digest that fragment before running. and then you can easily achieve the 3700bp fragment.

Try PFGE!! http://www.bio.davidson.edu/courses/genomics/method/pulse_field.html

You can also try a NuSieve agarose. Mix it with regular agarose in 1:3 ratio and run it under the same conditions. Be aware the NuSieve gels are more fragile! See if that helps

You can try adding synergel to agarose. It improves resolution of agarose gels. I'm not sure what concentration of synergel would work best for the separation of your fragments. I would start with 1% agarose and 0.2% synergel, and go from there.

Use 0.5% and run for 20v for 24hrs and see for a change but don't digest it.

I agree with Gabriele's suggestion. If you only need one of the two bands, it would be much easier to digest the unwanted fragment with a restriction enzyme that only cuts the unwanted fragment but not your desired fragment.

Why don't you try to use acrilamide? It's much more resolutive.

I agree with Sowersby's suggestion. You can use 0.6-0.7% agarose and run for 24h and 20V. You can also try with acrilamide but i don't think it wil be helpful enough

I believe Gabriele has given you exactly what is most practicable.

I know that very low conc agarose can be tricky to work with, and may still not separate your fragments. So, I would rather run another PCR with internal primers designed for the 300-400bp covering the area of the difference between the fragments. Otherwise, digest with an informative restriction endonuclease.

Also, it is important for you to consider the reason for the difference in the fragments: deletions? where? 5'/3'-end, or middle?

But be mindful that the best approach will probably depend on your plan for the fragments going forward.

Thank you all for your replies and suggestions.

I used a 1% agarose gel, around 25cm long, and ran it for 38h20m at 40V, and I managed to separate the two bands and purify the upper one.

I have done these types of separation very well, this is how it worked with me, run the gel as you describe cut out the two bands neatly as clean as you can, load these cut bands with agarose again in the wells for another round of gel electrophoresis. It works great. Using lower concentrations of gel is not good because the yield of getting the DNA lowers tremendously. In other words separation of DNA from agarose gets difficult no matter what you do. You might be good with 2 or at the most 3 agarose runs sequentially but it is outstanding clean DNA in the end. Hope it works for you.

The problem with trying to separate DNA fragments of >30Kb length in agarose is that DNA molecules that large are larger than the 'pores' in the agarose, even at low agarose concentration, so the DNA distorts, squeezing into the pore, and migrates faster than expected. This is why pulse-field gel electrophoresis was developed. I am not familiar enough with the conditions necessary to separate the fragment sizes you are interested in - typically this technique is used for chromosomal sized DNA, but I know it was worked out using sizes in the 30 - 100 Kb range.

Another suggestion would be to use low-melt agarose. I used to clone DNA out of that routinely. You use it at a lower concentration that regular low EEO agarose.

Use .5% gel and run at 80 volts for 3-4 hours. I used this method and think also best for you instead of long time run at low voltage........

Use 0.89 % of gel and run at 50 volt for 3 hrs.

Is that 3700bp or 37000bp?

0.8% gel can do the job. For Gel elution, see to it that load more pcr product which gives you pure dna for further processing.

I think you can use 0.6-0.7% agarose gel and increase voltage to 100 volt

2% agarose gel 80-100 volts for a few hours until well separated. I've used the method before to seperate two plasmid with similar size difference and it worked.

I suppose that you are running a digested vector. If so, you will know the exact sequence information and therefore it is very easy to isolate the desired fragment. The best approach is to digest the unwanted fragment with a restriction enzyme. You can use NEB's website to determine the right enzyme to use (http://tools.neb.com/NEBcutter2/). This way you can simplify the work, run the digest on a 1% agarose and purify the fragment.

TRY 1% AGAROSE AND RUN FOR AN HOUR OR TWO

Make 1.5 or 5% gel ,run at 60 volt.

I agree with Dongling Zheng, 2% agarose gel 80-100 volts for a few hours

Try 0.6% agarose and run at 30-35 volts for 1-2 hr, then speed up to 80 volts for 15 - 30 minutes or until they separated clearly

You could use pulsed-field electrophoresis

if you have the machine, it`s quite simple and would be one step. Of course, your DNA fragments are small enough to be separated in a normal electrophoresis as well.

I agree with Mary D Souza's suggestion to run two or three rounds of gels, to separate them. Digesting is not an option, if you want both fragments. Other useful method is to run column chromatography. But the DNA will get diluted and you need to precipitate again. You need to have reasonable amount of DNA to get the quantity you want after all the manipulations. Pulsefield is also very good option.

To separate two large fragments of similar size you DECREASE the concentration of agarose (to lets say 0.7%; in this way small fragments will stay more 'together', whereas bigger ones get more easily separated). To separate two small fragments (e.g. 80bp and 75bp) you do the opposite (increase to > 1%). If you do this (except for extreme situations) you can keep the same running time, voltage and gel size.

Because you cannot go much lower the 0.7-0.6%, the only further option is to make a longer gel and run longer. But 100 bp on a total of 3700 should be quite doable! Good luck

I think a 0.7% agarose gel run at most 30min can help for this separation

Why Cant you try 6% Native PAGE, it has quite better resolution then Agarose.

Check 0.8 or 0.7 / 0.75% agarose should work out well. Also try running it at a slightly lower voltage. Finally see that the buffer remains chilled throughout the run peiod.

The lowest Agarose concentration I've used was 0.3% (50 cm length) for Southern blotting and it worked well. (needed to seperate 10kb from 11 or so...), You can only run those gels at low speed; take TBE instead of TAE and run ON at 30 volts). In your case shorter gels should work, too if you'd use even 0.5%, but be sure to use high quality agarose!

Alternative: if you only need to purify one of the bands, then try to find a single cutter that cuts only in the fragment you don't intend to keep. Like this you can even run higher percentage gels...

In my opinion, I think the specificity of these primers are not high. And it might be those two genes are highly homologous. So try to redesign the primers!

Good luck.

If your 3600bp fragment has some restriction sites for particular enzymes, which aren't exist in another fragment, you would be able to digest that fragment before running. and then you can easily achieve the 3700bp fragment.

Does anyone here know of any papers that explain these various separation techniques?