---+ 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. Percentages ranging from 0.5-2% agarose can be used (lower percentages are better for separating larger products and higher is better for smaller products but 1% should work for most samples) For a standard 1% 50 ml gel: 1) Add 0.5g agarose and 50 ml TAE buffer to a 125ml flask and heat for 1:30 minutes (set up gel mold with appropriate well combs in the meantime). 2) After heating add 2.5 μl SYBR Safe (5μl SYBR Safe for every 100mL gel) and swirl to mix. 3) Pour the liquid from the flask into the mold and wait about 30 minutes for it to solidify. 4) Once the gel has solidified, remove the comb, loosen the gel tray and place the gel into a rig. Pour TAE 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 4°C fridge in the computer room adjacent to the gel area. The 1kb+ ladder is a combination 100bp and 1kb ladder which should be appropriate for any PCR sample (100bp ladders are quite useful for samples less than 1500bp, while 1kb ladders are best suited for larger samples). <img src="%ATTACHURLPATH%/10787018_1.jpg" alt="10787018_1.jpg" width='175' height='255.5' /> Basic Steps: 1) Load 6-7 μl of ladder into the first well (dye is already combined with ladder) 2) Combine dye and DNA on a cut out a sheet of parafilm: 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. 3) 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 Steps: 1) 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). 2) Set the voltage to 120V and run the gel for about 20-30 minutes (you can set a timer on the machine itself by clicking on the clock logo. The run will stop when the time runs out). It is advisable to check up on the gel from time to time to make sure that it is proceeding normally. 3) 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 Steps: 1) Place the tray in the imager and open the Image lab program. 2) Select Protocol 1, then Position Gel, then OK on filter 2, then center your gel. 3) Once centered, close the imager door and select Run Protocol. 4) Modify the image as you see fit.* 4) 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). If you want a physical copy you can also print the image. *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. ---+++ Gel post-staining Adding DNA dyes to agarose gels after electrophoresis (“post-staining”) can be advantageous when very crisp bands are needed (publication, low amount of DNA, etc). Adding nucleic acid dye to the molten agarose can cause slight inhibit DNA migration and bending of bands. Post-staining can reduce bending, increase DNA signal, and reduce background signal Steps: 1) after gel electrophoresis, place gel into small tray 1) add 15 µL of dye into 50 mL buffer (typically x1 TAE) and add to tray 1) gently agitate for 30 minutes 1) Remove buffer+dye 1) wash gel with ddH2O 2-3 times 1) image gel *Notes:* 1. 15 µL of GelRed into 50 mL buffer is the correct dilution 2. [[https://tools.thermofisher.com/content/sfs/manuals/sybr_safe_dna_gel_stain_man.pdf][SYBR-safe gel documentation]] notes that 50 µL of dye should be added to 50 mL buffer, though 15 µL of dye into 50 mL like likely sufficient (not tested) 3. Typically, dye is diluted in the same buffer that the gel is made of/was run in (e.g.: x1 TAE) 4. any volume of buffer+dye can be used, though it must completely cover the gel 5. Post-staining buffer can be reused up to 5 times by storing in dark storage container (e.g.: a 50 mL conical tube covered with aluminum foil)
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Topic revision: r3 - 2023-02-22 - MattMcGuffie