How to make an agarose gel

agarose gels are used in molecular biology to separate and purify nucleic acid fragments usually DNA of different sizes a dye is added to be able to see the fragments under ultraviolet light the samples are loaded into the wells of the gel and separated by size using an electric current applied across the gel in a running buffer DNA is negatively charged and it will make its way towards the positive electrode the smaller the DNA fragments are the easier would be for them to worm their way through the gel longer fragments will find this more difficult and will take much longer to make their way through the pores of the gel the size range of DNA fragments that can be separated by agarose in this way is around a hundred to twenty thousand base pairs agarose gels are made with varying concentrations of agarose depending on the size of the DNA fragments you want to separate or purify usually this is between Northpoint 4% and 3% if you want to separate or resolve large fragments then you will need to lower concentration of agarose so that the fragments can make their way through the pores in the gel more easily so it follows that if you want to separate small fragments then you will need a high concentration of agarose to make an agarose gel you will need agarose powder and a running buffer that you dissolve the powder into for a 1% agarose gel you would need one gram of powder in a hundred mils of buffer this will vary depending on the size of fragments you want to resolve the buffer is usually a Tris borate EDTA buffer children to tbe or twist acetate EDTA shortened to tae these two components are heated together to make the gel firstly make up the 1 times buffer you need using the concentrated stock we are using five times buffer so need to add 400 mils of water to 100 mils of concentrate after deciding the percentage gel you require then you need to weigh out the precise amount of gel powder you need in a volume suitable for the container or gel tray you're going to use the volume usually used is around 50 to 100 mils for smaller gels add the required volume of buffer to a suitable container 1 which will allow the liquid to expand when it's boiled and not to overflow carefully add the agarose and ensure that there are no lumps of powder before heating in the microwave place a lid or sponge bung loosely on the top do not tighten the lid this is very important as the liquid can become superheated the simplest way to heat in the microwave is in intervals of 1/2 a minute or so using safety gloves and goggles carefully swirl the mixture in between intervals to ensure that there are no particles of powder left these appear as tiny specks leaving a trail when the liquid is swirled it is important you don't shake the mixture as you don't want bubbles to form the liquid gel has to be completely clear with no gel specs before you proceed or your DNA will run through different concentrations of agarose in your gel and cause streaky results when the agarose is fully dissolved let it cool to 50 or 60 degrees centigrade this is hand hot and can be achieved by either placing in a water bath for 10 minutes or by carefully running under a tap we call the agarose to this temperature to prevent the perspex which is usually used for gel trays from being damaged once cooled add AI can be added to the gel to stain the DNA for visualization there are various dyes that can be used but the most commonly used is Athenian bromide care must be taken when using and disposing Ophidian bromide in both powder and liquid form especially for pregnant women it is a mutagen and possible carcinogen that can be breathed in and absorbed through the skin for this reason double gloves should be used and when making up a working solution of the etherium bromide do this in a fume hood stocks are generally 10 milligrams per milliliter and you will need 5 microliters of this stock per hundred mils of gel this will give you a final concentration of not 0.5 micrograms per mil in the gel if the din bromide is a DNA intercalate err inserting itself into the spaces between the base pairs of the double helix it yields low background and has a detection limit of one to five nanograms per band it is very important to remember to change your gloves after handling if they deem bromide without touching anything else in the lab this is to protect the safety of others by preventing contamination of equipment or surfaces that might be touched without gloves on remember that ethidium bromide will transfer to the running buffer so this must also be disposed of safely there are different types of gel tray some have ready-made ends to prevent the liquid gel pouring away while its setting others do not autoclave tape or masking tape can easily be used to seal the ends of these trays tear off a piece three or four centimetres longer than the end of the tray remember to turn over the end of the tape for easy removal once the gel is set very carefully put the tape in place at both ends making sure that there are no gaps for the gel to escape through while it's setting run your fingers over the edges to ensure a good seal you will need to pour the gel on a flat surface to make sure it's the same thickness across its length to form the wells that your DNA is going to be put into a comb needs to be inserted before the gel is poured the size of the comb you use will depend on the volume of the sample you have and the number of samples you want to run on the gel slowly pour your gel into your sealed gel tray when pouring it's important not to do it too quickly as bubbles and splashes can occur if bubbles do form then you can chase them to the side of the gel with the pipette tip where they won't cause any problems with the running of the gel this is important if bubbles form around the wells the gel sets in around 15 to 20 minutes and will change from transparent to more opaque remove the autoclave tape or the ends of the tray finally the comb can now be removed by putting it straight upwards it can be quite difficult to remove the comb but as long as the gel is completely set the well should still be okay you

Channel: Wellcome Connecting Science Courses and Conferences

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Keywords: agarose, gel, electrophoresis, DNA

Id: hcHXcH_oVLU

Length: 6min 59sec (419 seconds)

Published: Thu Jan 17 2013