I would suggest reading Brody et al. 2004 Biotechniques 37: 598-60. The suggestion some have made to run the gel at a lower voltage to minimize thermal diffusion of the small bands is only relevant if you use the usual (and not very good) electrophoresis buffers (TBE and TAE). What you really want is to run the gel as FAST as possible without the heating, and you just cannot do that with those buffers. They are really terrible for electrophoresis, because they are too salty (thus the thermal heating) and the contain Tris, which makes the problem worse, because Tris breaks down during the run and makes the conductivity problem and heating worse in a positive feedback loop.

By simply switching buffers, you can run very fast gels (say, 1000V or 100 V/cm). As Brody et al show, very small fragments (24, 40, 60 80, 100 bp), normally requiring polyacrylamide, could be resolved in1 mM lithium boric acid (or 1 mM sodium boric acid) in a 3% agarose gel in about 5 minutes (100 V/cm).  Make sure you use a good quality agarose with a low EEO value. You will also need to make sure you have reasonably low salt in your sample plus load buffer/dye. If your sample+loading dye is very salty, DNA migration will be retarded until the salts diffuse away.

Brody et al also have recommendations for the best simple salts and concentrations for separating other DNA size ranges (and RNA). But basically, either lithium (or sodium) boric acid in concentrations ranging from 1 mM to 10 mM will work for almost all your electrophoresis needs. You will save a lot of time on electrophoresis and on buffer preparation, as well as money, because the solutions are simple, low concentration, and cheap. You can buy the concentrated electrophoresis media and loading buffer pre-made (http://www.fasterbettermedia.com/) or you can make it yourself. (Note: I have do not have any financial stake in this company)

For an enlightening history of electrophoresis media see also Brody and Kern 2004 Anal. Biochem 333: 1-13. It is fascinating to me that TAE and TBE have remained so common when they are so poor at the job they are supposed to do. An illustration of the power of tradition and the inertia that can take hold in our routine work.

Hi there!  You would need to increase the percentage of the agarose gel that you are using.  Generally, to separate smaller DNA fragments, you need to run them on higher percentage gel, whereas larger DNA fragments require lower percentage gel.  It may be best to consult a Molecular Cloning Manual, but I would expect that you would need at least a 2% gel or maybe higher in order to reliably separate out the bands (especially the 41 bp).  Finally, since the bands that you are trying to resolve also are close to one another in size, you would need to run it the gel for a long time at lower voltage (maybe 60 V instead of 100 V)to ensure separation (I am not sure if you will ever be able to resolve the 175 bp band from 169 bp band - I guess if you see two bands very close to one another at the right size, you can assume that one is 175 bp and the other 169 bp).  Hope that helps.

You will need to run a 6% acrylamide gel to resolve those small fragments with any sort of confidence. Run a DNA ladder with low molecular weight fragments similar to the sizes you expect. After running you can stain the gel with ethidium bromide or some other stain to visualize the fragments.

Good Luck!

I agree with both of the above but if you must run agarose then run the gel very slowly to minimise thermal diffusion of the bands and preferably in a cold room if one is available. Use 2V /cm of gel length not the usual 5V/cm . If no cold room available then put the gel and the buffer in a fridge before use may help a little

you can use the principle and technique based on which samples are run on an sequencing gel where we can separate DNA fragments of even 1bp difference. GOOD LUCK.

Usually, Tris base, Acetic acid, and EDTA (TAE) buffer is used when the DNA fragment is greater than 15000bp, otherwise, TBE (Tris/Borate/EDTA) is more appropriate. When DNA fragment is less than 1000bp, TBE would yield high resolution. I suggest that you prepare 1x TBE buffer instead of 5x TBE buffer, and then run the gel for a longer time.

Goodluck.

I would suggest reading Brody et al. 2004 Biotechniques 37: 598-60. The suggestion some have made to run the gel at a lower voltage to minimize thermal diffusion of the small bands is only relevant if you use the usual (and not very good) electrophoresis buffers (TBE and TAE). What you really want is to run the gel as FAST as possible without the heating, and you just cannot do that with those buffers. They are really terrible for electrophoresis, because they are too salty (thus the thermal heating) and the contain Tris, which makes the problem worse, because Tris breaks down during the run and makes the conductivity problem and heating worse in a positive feedback loop.

By simply switching buffers, you can run very fast gels (say, 1000V or 100 V/cm). As Brody et al show, very small fragments (24, 40, 60 80, 100 bp), normally requiring polyacrylamide, could be resolved in1 mM lithium boric acid (or 1 mM sodium boric acid) in a 3% agarose gel in about 5 minutes (100 V/cm).  Make sure you use a good quality agarose with a low EEO value. You will also need to make sure you have reasonably low salt in your sample plus load buffer/dye. If your sample+loading dye is very salty, DNA migration will be retarded until the salts diffuse away.

Brody et al also have recommendations for the best simple salts and concentrations for separating other DNA size ranges (and RNA). But basically, either lithium (or sodium) boric acid in concentrations ranging from 1 mM to 10 mM will work for almost all your electrophoresis needs. You will save a lot of time on electrophoresis and on buffer preparation, as well as money, because the solutions are simple, low concentration, and cheap. You can buy the concentrated electrophoresis media and loading buffer pre-made (http://www.fasterbettermedia.com/) or you can make it yourself. (Note: I have do not have any financial stake in this company)

For an enlightening history of electrophoresis media see also Brody and Kern 2004 Anal. Biochem 333: 1-13. It is fascinating to me that TAE and TBE have remained so common when they are so poor at the job they are supposed to do. An illustration of the power of tradition and the inertia that can take hold in our routine work.

Hi, you got very good advice here.

One thing is still missing, though: titrate different amounts of sample to load in the gel. Make sure that you have an adequate amount to see thin, sharp bands as opposed to thick, smeary ones.

As suggested before, trying different buffers can be beneficial but if you can't do it then just running the gel at slightly lower Voltage (80V if you usually use 100V) to prevent heating or run the gel in the cold room. Using a 2% gel should help you get a better resolution. Make sure agarose is completely dissolved before casting the gel (particularly important with high percentage gels).

Good luck with your experiment and keep us updated so that we can keep helping you.

Best,

P.

Hi there

I think you can try following

1. increase the conc. of gel i.e. at least 3%

2. use 1X TBE

3. Run the gel at 50 -60 V for 1.5 hours, use 25 bp ladder (e.g. HyperLadder™ 25bp of BIOLINE) or 10 bp ladder (e.g. 10bp LowRange DNA-Ladder of Geneon)

hope this will help.

Actually you should not use agarose in more than 1.5%. In your case I suggest that you run PAGE.

Yes, acrylamide gels are the best choice for your separation. 

Set the volume of buffer TBE 0.5x has 75 ml and do vary the quantity of agarose. You can try a gel of 1.5% with 1.125 grams of agarose and 75 ml of 0,5x TBE buffer. To migrate for 60 minutes a 100 Volt.

Good luck

I add 1 gr from agarose to 100 ml of TE buffer 10x and  100-120 V at 30 min

Hi,

I think 1% agarose in 1X TBE should work well at 80 volts.

low the voltage, and put it for more time, try using 2.5%, and don´t use ethidium bromure with it because i will go out of the gel, so use sybr green, and good luck with that. if it doesnt work try doing an acrilamide gel for running your DNA samples

http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_BenchGuides_SourceBook_Section_VII_-_Separation_of_DNA_in_Polyacrylamide_Gels.pdf

Hi there

Any band less than 70 bp usually does not resolve very good at agarose gel for these size of bands SDS PAGE is recommended. 

Hope it will help you

using 60V at 2% agarose give good result in this case

using 70v at 2.5%agrose and 45 mintgive good result in this case

Agarose gel preparation was performed 1% concentration was prepared by suspending 1g agarose powder in 100 ml TAE buffer. The electrical power was turned on at 1 watt for 65 min.