Hi,
In the previous post I explained how to quantify DNA, and now we can find the molecular weight of that extracted DNA.
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.
Clear now?
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.
| 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.
Clear now?
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.
Well, choosing the percentage of agarose has more to do than just thet things explained. As you mentioned, bulkier DNA cant move easily through very small pore size (pore size decrease with increasing agarose %). But, in some cases, where you want to resolve very close sized DNA molecules (say one sample is 100 base pair long and another is 150 base pair long), you should choose higher % agarose gels. You can go easily upto 2%. Lower the agarose %, more fragile the gel is and difficult to handle.
ReplyDeleteThanks for sharing Mr.Venkat. Actually I had never used gel for resolving DNA with nearly equal size. Got your point. Thanks. I updated the post. Hope now it's correct?
Delete@Kanmani: You are welcome :) Its very nice to come across a blog with great info about biotech stuff, I got to know some new details.
ReplyDeleteTo add a little more on the information you have here, EtBr is very toxic (also carcinogenic) and though they are still used in labs, there are some other alternatives which are considered more safe than EtBr. SYBRgold or SYBRgreen from Invitrogen are commonly used in our lab for post staining (staining after running the gel) and some pre-staining dyes (mixed to the agarose gel before setting the gel) are also available. These dyes are less toxic and non-carcinogenic.
Oh, yeah, EtBr is carcinogenic I know, We have to handle it with care. Oh, this is the first time I'm hearing about this SYBR, but, how about the cost? And, you are a biotechnologist?
DeleteI would like to have you in my tech-link, if you don't mind, could you please drop a mail at kabc1993[at]gmail[dot]com ?
@Kanmani: SYBR is a commercial product from Invitrogen (A popular company for fluorescent markers and other biotech stuff). Price for a 500ul unit should be around 5000 rupees. This is in 10,000X concentration, so can be used for around 100 gel staining. May be more expensive than EtBr, but much safer product I believe. There are other products from different companies, can be chosen according to one's needs.
ReplyDeleteNo, I am a nano-biophysicist. I use micro-nano fluidic devices to do single molecule studies of biomolecules using different methods.
Sure, I ll :)
Have a good day!!!
Oh, fine. Thank you.
Deletegood day!