Gel electrophoresis
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- Project Due Date: 3rd may 2012
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Gel electrophoresis
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 !
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Re: Gel electrophoresis
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
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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- 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
Re: Gel electrophoresis
thanx alot !!