Hi all,
I have to run a gel to separate small fragments, 50-25 bp..What are the best running gel conditions (in terms of time and voltage)? I did not understand very well how you choose the time and voltage depending on the size of the fragments you want to visualize. Last time for example, I tried to run a gel to separate bands of a 25 bp ladder . I run it for 3 hours at 60 V, but after that, I saw that the bands had not been separated yet. Does this mean that generally you have to increase your run time as the size of the fragments gets smaller?Is there not risk that your gel is too short to separate those bands? And what if the fragments are very large? I am confused.
Thanks a lot
Hi Silvia,
Generally, the smaller the fragments you want to distinguish, the higher percentage gel you want to run. Resolution power isn't really influenced (that much) by voltage so you have some freedom to kick the voltage up and still have the ability to distinguish 25 and 50bp bands. The larger the fragments, the smaller the percentage gel you will need to get them into the gel. Generally, for anything 1KB or larger, i use a 0.7% agarose gel. For small fragments, I use gels that range in percentage from 3 to 4% agarose, however you will need a special additive or other types of agarose that allow for higher percentage gels (Nusieve, etc.). You will need to experiment with voltage and times with respect to different percentages as the run times and resolution power will change with the change in percent agarose.
Hi Silva,
My recommendations would be that you use a PAGE gel, These are ideal for the separation of sizes below 100bp.
However, if you want to use agarose type gels then I have found that running them at 150-200V for about 30 mins run in a cold room works well for separation of small products, The reason for this is the high voltage keeps the bands sharp for distinguishing the difference of similarly sized dna molecules!
Hope that helps?
Hi Silvia,
I m using Nondenaturing Nucleic 7 Acid PAGE Biorad.
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_1658100.pdf
for less than 100bp do PAGE, over that (100-500bp) you can use TBE agarose gel then for more higher bp products use TAE agarose gel for better product visibilty.
Jobin has a good point. Chose the most appropriate gel type first then change the concentration of the gel matrix if required for better resolution. I have not used polyacrylamide for DNA samples but it should work fine, if not better too. Previously, in order to separate DNA samples of 50bp, I used a TBE 2% agarose gel and ran it at 60V for 45 minutes.
In general, the higher the voltage, the higher the "driving force" provided to separate the DNA fragments (mobile phase) across the gel matrix (stationary phase). So higher voltage makes bands travel faster reducing the run time required. Both short and long bands are affected although not in a linear manner. As a net effect, the increase in migration speed of shorter bands (1000bp) when the same voltage increase is applied.
The easiest method of optimising your gel run is by using a specific DNA ladder (ie. for 50bp samples, use a DNA ladder that has a range of ~20bp -1000bp) and stop the gel run as soon as the first band reaches the end of your gel. Always make sure that all bands are still in your gel to confirm that there are no "over-runs". There is always a possibility that the band that you are interested in running out of the gel and into the running buffer if the running time is extended too long.
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