Gel Electrophoresis

In order to analyze PCR results, the products are run on an agarose gel, which is then analyzed in UV light to ascertain the length of DNA fragments in the PCR reaction.

Making the Gel

A standard 1% agarose gel uses 1g of agarose for every 100 ml of buffer. A different percentage may be used, and gels with less than 1% agarose may be used to clearly distinguish products of very similar sizes. For a standard 50 ml gel, add .5g agarose and 50 ml SB buffer to a 125ml flask and heat for 1:30 minutes. Meanwhile, assemble a gel tray into its holder and find a comb with an appropriate number of wells, then place the comb into the rig. After heating add 2.5 μl SYBR Safe (5μl SYBR Safe for every 100mL gel) and swirl to mix. Pour the liquid from the flask into the rig and wait about 30 minutes for it to solidify. Once the gel has solidified, remove the comb, loosen the gel tray and place the gel into a rig. Pour SB buffer until the gel is thoroughly submerged.

Loading the Gel

DNA samples are loaded into the wells of an agarose gel using a p20 or p10 pipettor. First, gather all PCR products that are to be run, an appropriately sized ladder, and 6x loading dye. The latter two may be found in the 4C fridge in the computer room adjacent to the gel area. 100bp ladders are quite useful for samples less than 1500bp, while 1kb ladders are best suited for larger samples. First, load 6-7 μl of ladder into the first well. The easiest way to combine dye and DNA is to cut out a sheet of parafilm and make a drop of 1 μl of dye onto the parafilm for each sample to be run. Next, add 5 μl of PCR product to the dye and pipette up and down to homogenize. Once all samples are combined with dye, load them into the gel, making note of what sample goes into what lane.

Variation: Instead of combining all of the samples and then loading them, it is possible to load each sample as its combined with the dye. To do this, pipette up and down as normal, then push the pipettor a little bit past the first stop, enough to suck up all of the combined sample and dye. Now load this into a well, taking care to avoid shooting too much air into the well, as this may displace the sample. This method minimizes the amount of time samples spend exposed to the air, and thus prevents them from partially drying out on the parafilm.

Note: Close PCR tubes when not being used. PCR products tend to dry up when exposed to air, leaving a lower volume that has a higher concentration of DNA.

Running the Gel

Once all samples have been loaded, attach a lid to the rig, and attach the lid to the BioRad power supply (NOTE: always make sure that the current is off or paused before inserting or removing a cords from the power supply). Set the voltage to 150V and run the gel for about 30 minutes. It is advisable to check up on the gel from time to time to make sure that it is proceeding normally. When the gel seems to be completed, pause/stop the voltage, disconnect and remove the lid, and take the gel (in its tray) to the Bio-Rad gel imaging machine in the adjacent room.

Imaging and Analyzing the Gel

Place the tray in the imager and open the Image lab program. Select Protocol 1, then Position Gel, then OK on filter 2, then center your gel. Once centered, close the imager door and select Run Protocol. Once the imager finishes analyzing the image, save the image in Documents/[your name]. Note that the program saves files as .scn, which can only be opened by Image Lab and related programs. For a simpler version which can be viewed from any computer, select snapshot, then save a common file type (ex: .jpg). Emailing the images to oneself is a useful way to keep track of them.

Image lab contains several tools to aid in analyzing the gel image. Hitting the change contrast button allows manipulation of the brightness of the image, which may be helpful to clearly see faint bands. By clicking Lanes and Bands, then Automatic, the program puts labeled lanes where it perceives them. Bands can similarly be applied to the image.

When done modifying the image, print it out (saving it again / taking another snapshot might also be a good idea) and paste the picture in a lab notebook.

-- Main.AurkoDasgupta - 17 Jun 2011

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Topic revision: r1 - 17 Jun 2011 - 22:08:59 - Main.AurkoDasgupta
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