|Agarose gel electrophoresis|
But, how? Simple, you need to run an agarose gel!
Preparation of agarose gel:
Agarose gel must be prepared based on the DNA we are going to run. For example: if you are going to run Genomic DNA, you must use 0.6%gel, for plasmid DNA, you must use 0.8% gel, for PCR(Polymerase chain reaction) sample, you must use 1.2% gel.
You may ask, why three different % of gels? The answer for your question is:
Genomic DNA will be bulkier than pDNA - so, we must use a gel with less percentage, so that the pore size will be greater and the genomic DNA can move easily.
In case of PCR sample, the gene amplified won't be bulky, so, it moves fast, very fast in low percent gel, that's why to have an optimum speed we use a higher percentage of gel comparatively.
And, when our aim is to separate two sequences with relatively equal size, we must use higher percentage gel, this increases the run time, but, still we can effectively resolve two sequences with more or less equal size, say, 500bp and 550 bp.
Okay, how to prepare the agarose gel? It's very simple, just weigh 0.8gm of agarose and dissolve in 100 ml of TBE buffer. Microwave this until the agarose gets dissolved and you get a clear solution.
To this clear solution, add EtBr (Ethidium Bromide) 3micro liter for 25 ml (Note: while adding EtBr, the solution should not be very hot, it must not be very cool- if it gets so cool, it'll solidify, the solution must be of bearable warmth). Now, after adding EtBr, pour the gel in the gel cast with comb and allow it to solidify.
After it solidifies, add TBE buffer over it (for lubrication) and remove the comb carefully. Lanes will be formed.
Place the gel in the Agarose gel setup with anode and cathode. Load your sample DNA* in the lane.
Load a standard DNA ladder (for finding the molecular weight of your sample DNA.) And run at 75 V. Stop when the sample reached 3/4 th of the gel.
Now, view the gel under UV platform, the DNA band will fluoresce due to the presence of EtBr in the gel. Measure the distance travelled by your sample from the lane in cms. Also measure the distance travelled by the standard ladder.
The standard’s molecular weight is known already. So, plot a graph with Molecular weight of the standard in X axis and Rf value (Distance measured in cms) in y axis.
From the standard graph, by plotting the rf value of our sample, its molecular weight can be found.
*Preparation of DNA sample:
Take 5 micro liter of sample in an eppendorf add 5micro liter of tracking dye to it. Give a brief spin in centrifuge for mixing them.
Tracking dye contains Glycerol- which gives density to the sample, so that it goes down into the lane easily while loading; it also contains bromophenol blue which acts as a visualising agent while you are running the gel.
Got it now, any doubts? feel free to comment, I'l try to answer as soon as possible.