Hi Mustafa,

use a 0.7 % agarose gel (better separation of DNA molecules of larger size) and let the gel run slowly (12V per cm or even less --> dependent on the size of your gel). And of course, let your gel run long enough.

Then you should separate them easily.

Best,

GAD

Hi Mustafa,

use a 0.7 % agarose gel (better separation of DNA molecules of larger size) and let the gel run slowly (12V per cm or even less --> dependent on the size of your gel). And of course, let your gel run long enough.

Then you should separate them easily.

Best,

GAD

Which buffer are you using? TAE (instead of TBE) would be the better choice.

see: http://molecularstation.com/agarose-gel-electrophoresis

I've had the same problem sometimes. What I usually do is to look for a restriction enzyme that cut the fragment that I'm not interested. After a digestion with that enzime you'll be able to cut the band from the gel.

Mustafa,

try using a lower agarose concentration of probably 0.6-0.7% and run it as slow as possible to get better separation, somewhere on the 60-70V range should do the trick. it's going to run slower, but it's gonna do the trick.

Best of luck

Thank you very much Guido Drexler.

Actually, I've tried to run it as long as I could, but that didn't help. I'll try 0.7% and slow run together to see if that help.

Thanks again.

Mustafa

Thanks a lot Paul Lesbats for providing this wonderful table. I haven't thought about having a gel less 0.5%.

Thanks again.

Mustafa

Thanks a lot for everybody. Your information is valuable and helpful for sure.

Mustafa

Use a .7% gel

As the others... use a big gel (if it is possible) and/or use 0.5-0.6 agarosa gel. Less is possible but you could have some problems to manipulate it (it could be broken). Don't load too much amount of DNA: bands will be thicker and they will be closer.

Luck!

Use a long agaros gel (0.6-0,7%) and let it run for a longer time to get a better separation. However, I'd suggest to run it on a higher voltage (e.g. 105V, without melting it) - slower voltage takes more time, resulting in more diffusion of the bands, ending up in worse separation. I don't understand why running the gel extra slowly should improve the quality anyhow?

Of course, cleaving one band with a restriction enzyme is a good trick to shift it!

If you still can't separate them - are you sure restriction worked properly? Maybe a problem there?

All ideas are great- try to combine them. Use lower gel concentration, try long gel, run it slowly and try to put less DNA on it. Maybe it helps, it usually does. Good luck!

0.5- 0.7% gel, 60V and low amount of DNA, usually works.

Good luck!

These suggestions should take care of it, but if they don't you could consider pulsed-field gels (require a special apparatus). They greatly increase the resolution of larger DNAs. I would also mention that surprisingly, the smaller band is always slightly contaminated by the larger band(s) on an agarose gel, so you need to keep that in mind is you are subcloning etc..

You can get a separate band for 8000bp DNA. I would never recommend using a lower concentration of Agarose. I would say lower the current and try. You may need to standardize this electrophoresis for your need. According to me 1% agarose is a fairly good concentration and lowering it further may not be of any use.

Hi Mustafa

Try the band stab PCR technique described by Bjourson and Cooper (Nucleic Acids Research 20 (17) 4675, 1992). It may solve this and many similar problems.

Best Wishes

Chris

Thanks Chris Shaw. I will.

I always use .7% gel and run at 70V....with me that always does the trick. Good luck!

Ues 0.7% gel and run at 70v. This works for me. Good luck

1% gel is good to separate in the range of 500- 2000bp, 0.8% handles 1000-4000bp well, but you can go even as low as 0.5% for fragments you describe... Run the gel slowly as suggested before, check if you use TBE or TAE buffer, TBE is much better for sharp bands. It is even more important to avoid overloading the gel, so if your bands are not sharp lines, it will be much harder to separate. You may also consider not purifying the fragment and ligating a mixture (12000 bp and 8000 bp), sorting out the right recombinants later. I'm not sure what you want to do in the end, but the larger the fragment the lower the yield from a gel-extraction and the higher the chance for mutations. Is the 12000bp band a plasmid backbone (a very large fragment) or is it something else?

Yes Jürgen, the 12K is the plasmid backbone.

Thanks for your comment

as suggested by others try to use lower percentage for your gel, run at lower voltage and load less DNA on the gel. I have attached a guide that could be useful... 

Best, 

Dacquin

http://www.genomics.agilent.com/files/Mobio/Nucleic%20Acids_Gel_Electrophoresis.pdf

Totally agree with Pablo García-Miranda. He was pointed out the most appropriate way.