Gel electrophoresis

AFTER you've done your research and concluded your experiments, it is time to prepare for the science fair. Ask specific questions about preparing for a science fair, including how to set up your display board, how to prepare a presentation, etc. (Please post questions about selecting a project or conducting your experiment by posting in the appropriate "area of science" forum.)

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Gel electrophoresis

Postby kool.boy.ameer.ahmad » Tue May 01, 2012 8:27 pm

can i make the gel on stove !! ive tried microwave n stove both ! non worked :( i dont know whats wrong ! can u please tell for how long did u put the mixture in microwave n in how long did it gel !!! n 1 more question !! i think i m using the wrong agarose powder ! can u tell the xact information of the powder which u used ! whats its chemical fomula n whats its color n all !!! AND CAN I USE GELATINE !??? thank u so much ! im going to do this project in my science fair !! :) plz reply fast !
kool.boy.ameer.ahmad
 
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Re: Gel electrophoresis

Postby jmehta14 » Wed May 02, 2012 11:55 am

Dear Kool Boy,
From the site listed below
http://www.methodbook.net/dna/agarogel.html

Making the gel (for a 1% gel, 50 mL volume)

Weigh out 0.5 g of agarose into a 250 mL conical flask. Add 50 mL of 0.5xTBE , swirl to mix.

It is good to use a large container, as long as it fits in the microwave, because the agarose boils over easily.

Microwave for about 1 minute to dissolve the agarose.

The agarose solution can boil over very easily so keep checking it. It is good to stop it after 45 seconds and give it a swirl. It can become superheated and NOT boil until you take it out whereupon it boils out all over you hands. So wear gloves and hold it at arms length. You can use a bunsen burner instead of a microwave - just remember to keep watching it.

Leave it to cool on the bench for 5 minutes down to about 60°C (just too hot to keep holding in bare hands).

If you had to boil it for a long time to dissolve the agarose then you may have lost some water to water vapour. You can weigh the flask before and after heating and add in a little distilled water to make up this lost volume. While the agarose is cooling, prepare the gel tank ready, on a level surface.

Add 1 µL of ethidium bromide (10 mg/mL) and swirl to mix

The reason for allowing the agarose to cool a little before this step is to minimise production of ethidium bromide vapour. Ethidium Bromide is mutagenic and should be handled with extreme caution. Dispose of the contaminated tip into a dedicated ethidium bromide waste container. 10 mg/mL ethidium bromide solution is made up using tablets (to avoid weighing out powder) and is stored at 4°C in the dark with TOXIC labels on it.

Pour the gel slowly into the tank. Push any bubbles away to the side using a disposable tip. Insert the comb and double check that it is correctly positioned.

The benefit of pouring slowly is that most bubbles stay up in the flask. Rinse out the flask immediately.

Leave to set for at least 30 minutes, preferably 1 hour, with the lid on if possible.

The gel may look set much sooner but running DNA into a gel too soon can give terrible-looking results with smeary diffuse bands.

Pour 0.5x TBE buffer into the gel tank to submerge the gel to 2–5 mm depth. This is the running buffer.

You must use the same buffer at this stage as you used to make the gel. ie. If you used 0.6xTBE in the gel then use 0.6xTBE for the running buffer. Remember to remove the metal gel-formers if your gel tank uses them.

Also try reading this site to help.
http://www.mcdb.lsa.umich.edu/labs/madd ... _gels.html


I hope this helps!
Jay Mehta
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Re: Gel electrophoresis

Postby kool.boy.ameer.ahmad » Wed May 02, 2012 12:27 pm

thanx alot !! :)
kool.boy.ameer.ahmad
 
Posts: 2
Joined: Mon Apr 30, 2012 11:02 am
Occupation: student Grade 9
Project Question: science- Biotechnology
Project Due Date: 3rd may 2012
Project Status: I am conducting my experiment


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