I apologize if this is a basic question. I had a hard time finding answers to it.
I try to deepen my understanding of gel electrophoresis and wonder how should I change the voltage and run time in gel electrophoresis when I go from small gel to medium or large?
according to this website:
https://www.thermofisher.com/il/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/na-electrophoresis-education/na-electrophoresis-workflow.html
"if I run DNA fragment that is less then 1kb I should use voltage between 5–10 V/cm.
Voltage to be applied (V) = distance between the electrodes (cm) x recommended V/cm."
When I measure the distance between the electrodes should I measure from the start to the end of the tray? or from the start to the end of the gel? this will give me very different sizes.
let's assume that I measure from the start to the end of the gel and got the following sizes:
the length of the small gel is 10cm
the length of the medium gel is 14cm
the length of the big gel is 17cm
let's assume that I decided to use a 7V/cm voltage.
does it mean that in the small gel I should use:
V = 10cm x 7V/cm
V = 70.
And in the medium gel 98V and in the big gel 119V?
Did I get it right? Or am I missing something basic here?
What about running time? Assuming I change the voltage according to the size of the gel, should I change the running time? If so, how should it be changed?
Your queston is about agarose gel or PAAG?
Your basics is ok, now you need just practice, and do not afraid to make a mistake)))
Your queston is about agarose gel or PAAG?
Your basics is ok, now you need just practice, and do not afraid to make a mistake)))
Voltage calculations are made from the distance between the platinum wires at the bottom of the gel box. As for the time you run the gel depends on the size of the gel. 100 bp runs at about 1 cm hour per volt/cm using half strength TBE. So at 10 volts/cm it will run 10 cm but it is best to use dye visualization and stop when bromophenol blue has run 3/4 up the gel. I think it is important to use fresh running buffer (changed daily) because when there is a difference between the conductivity of the gel and running buffer leads to inconsistent results.
- The above formulas and your rational is correct but perhaps you are over thinking this somewhat:
- Most gels can be run for 30 minutes when very small to 1 hour @ 80V to 120V:
- Just keep an eye on your dye front and make sure it is no more than 2cm from the bottom for a 1 hour duration: for small and medium sized gel tanks @ 1-2% this tends to be 1 hour to 90min of running time
- I tend to pay attention to these formulas more when I am running over night gels
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