Hi Camilo! The ladder works well in a gel of 0.8%. As far as it concerns the voltage to use and the time it takes, you have to consider the distance between the electrodes of the apparatus and the size of your fragments. For fragments 1-12 Kb use a voltage of 3-6 V/cm, for >12Kb use 1-2 V/cm.

Of course lower is the voltage, longer is time required. The electrophoresis should be stopped when brophenol blue run about 2/3 - 3/4 of the gel length. For example in my apparatus with the distance between the electrodes of 15 cm and with a gel length of 11 cm , a run usually takes 1h with a voltage of 75V.

Good luck!

Hi Camilo! The ladder works well in a gel of 0.8%. As far as it concerns the voltage to use and the time it takes, you have to consider the distance between the electrodes of the apparatus and the size of your fragments. For fragments 1-12 Kb use a voltage of 3-6 V/cm, for >12Kb use 1-2 V/cm.

Of course lower is the voltage, longer is time required. The electrophoresis should be stopped when brophenol blue run about 2/3 - 3/4 of the gel length. For example in my apparatus with the distance between the electrodes of 15 cm and with a gel length of 11 cm , a run usually takes 1h with a voltage of 75V.

Good luck!

Hi Camilo, in a mini-gel (7 cm long) I electrophorese at 100 volts (constant) for about 20-25 minutes. This is approximately the time it takes bromophenol blue to traverse about 2/3 of the gel's length. It works well. Good luck.

If better resolution of bands is desired then try to use TBE buffer instead of TAE to run your gel and also try to run a 15 cm gel.

Yes.. TBE buffer is best for good band separation. For voltage setting you have to consider the distance between the electrodes in the electrophoresis tank (2V/cm is good) . Time will be based on the voltage and gel length.

We use standardly 120V with different systems. The gels are usually like 10-15 cm long and the chamber (electrode distance) is about 20-25 cm, I would say.

I routinely use TAE but at double the concentration (2X instead of 1X). If you need good separation, prestain your agarose gel with GelRed (see for example http://www.biotium.com/product/product_info/newproduct/gelstains.asp) and monitor separation of DNA bands as the gel electrophoresis progresses. In my opinion 1% gel should be just fine. Run it for shorter or longer period of time depending upon what separation you need to achieve. Of course the longer the gel the better, however if you run for too long the ions in the buffer get depleted, which has its negative consequences.

Hello dear Camilo, i am using 1% agarose gel in 1X TBE buffer (Cool) at 100V for 25-30 min and Get good separation of bands.

Hello Camilo

in my opinion I think it will be impossible to separate bands with low length (250 bp) and high lenght at the same time with the same conditions. Do not try to get a resolution of your ladder as fermentas show you in the users manual, all you have to do is work at low concentrations of agarose for high lenght bands (0.5 -0.8%) and at high concentrations of agarose for small sized bands (1-1,5%). This way you will get a good resolution for your ladder in the range you want to analyze.

regards

and good luck!

And work with low concentration of etBr or gelred (or whatever) for minimize background.

I'm going to give the layman's answer here (though I usually don't) and recommend you just load your marker in two wells - one in a small quantity so you can see the larger bands well, and one in double the quantity so you can see the smaller bands well. And then run slooooow. And maybe consider staining with EtBr after the electrophoresis instead of including it in the gel. It depends on how important your experiment is - after all there's a huge difference between "check your construct" and "make figure for paper".

Hello Camilo,

in the lab we used gels of 7cm, 1% agarosa, and use 100v during 30 min.

As the good old Molecular Biology (Maniatis) stated 5 Volts/cm (cm of distance between electrodes) will give you the best resolution. TBE has a higher buffering capacity than TAE (so if you use TAE avoid minipreps with small volume cuvetters since you will loose the buffering capacity). If you use large cuvetes you can use TAE.

If you are in a rush and want to use higher voltage to run the gel, you can always put the cuvete on ice or even in the cold room and go to double vots/cm

You should use TBE instead of TAE and then you should run the 0.8 % gel at around 45 to 50 Volts it will take longer time such as 4 to 5 hrs but it will give you distinguish band of the marker............ also you have to take care the amount of marker you should add in the well ...............

In our lab, we usually use 0,8% agarose gel in 1X TBE buffer and use100V for 30 min or more, depending the size of your fragments and the gel length. Try not run to much, so you may get a good resolution of the band. Good luck!