how do you determine the correct voltage and time of running of an agarose gel? - (Dec/16/2005 )
Hello,
I am taking a molecular technique's course, and one of the questions on our practice exam is: List the appropriate electrophoresis conditions (% agarose in gel, voltage, approximate time) for the products below:
50 and 150 bp restriction digest
genomic extract
1400 bp PCR product
I found the % agarose in a published chart, but I'm not sure about the voltage and the approximate times. Doesn't it depend on what size our gel is and how thick we pour it? Is there a generic voltage and time that is usually used for these fragment sizes? All I could find in various resources is that theoretically a voltage of 1-5 volts per centimeter should be applied when running an agarose gel ...
Thanks!
Sonia
I am taking a molecular technique's course, and one of the questions on our practice exam is: List the appropriate electrophoresis conditions (% agarose in gel, voltage, approximate time) for the products below:
50 and 150 bp restriction digest
genomic extract
1400 bp PCR product
I found the % agarose in a published chart, but I'm not sure about the voltage and the approximate times. Doesn't it depend on what size our gel is and how thick we pour it? Is there a generic voltage and time that is usually used for these fragment sizes? All I could find in various resources is that theoretically a voltage of 1-5 volts per centimeter should be applied when running an agarose gel ...
Thanks!
Sonia
As a thumb rule, higher voltage/gel percentage and lesser time is used for smaller products and the reverse for larger products. For 50-150 bp I generally use something like a 2% gel, run for around 30 minutes at 90-100v. I run my genomic DNA in an 0.8% gel at 25-30v for many hours. I find anything above 40v to cause erratic run of genomic DNA.
For a 1400 bp product, 1-1.5% gel at 70-90v for 30-40 min would be just fine.
I usually keep the thickness of my gels low (~4mm) as thick gels obscure faint bands. Also never add more than 2-3mm buffer above the level of the gel as in long runs it might cause a 'sliding effect' of bands.
It's not the voltage that's important, it's how many volts per cm between the negative pole and the positive one that's important (as sonia stated). It also depends on your application. If you want do determine if a pcr was positive, you need to know wether your product was formed, so you don't need to know exactly how big it is (if you know it's there it's good). If you however need to do a gel extraction and the different parts aren't too different in size, you need a very good separation...
Here's some examples of what I do...
For me, it depends, when going for small fragments (up to 500 bp), I usually go for a 3-4% gel and apply roughly 3-4 volts per cm and let it run for an hour or so (this gives me a good separation of the ladder I'm using which is roche's ladder five). When doing bigger parts, up to 5 kb, I take a 1% gel and apply 4-5 per cm volts for 45-50 minutes.
When doing gel extraction of for instance a cut vector which gives fragments of 4 and 7 kb, I go for .7% gel and put it on 3-4 volts per cm and let it run for 2 hours or more (you need a bigger gel system).
Yea, gel running voltage depends on whether you just want to check the band, or you want a good separation for further study. Higher voltage on a lower percentage agarose gel will run faster, but the resolution of bands is not as good as lower voltage on a higher percentage gel. Another thing is, too high a voltage may cause the gel to melt; whereas too low a voltage may cause the diffusion of the product that you loaded into the gel.
Good luck.
Hmm I am having a similiar problem in determining as well...I used 1% agarose for a prodct of 570bp, running at 120V for 50mins but I didn't get any bands at all...what changes can be suggested for me? Thanks!!
Run a DNA ladder alongside your sample. If it turns out fine it is a problem with your sample e.g. no amplification of your expected PCR target
The ladder is fine but if there's no amplification shouldn't there be bands of the primers? I dun get any bands at all and was thinking the product has been wash-out due to not suitable gel running conditions such as volts, time and percentage...