Peccoud lab Protocol: Visualizing DNA following size exclusion on an agarose gel
In agarose gel electrophoresis, a sample of a biological molecule, in this case, DNA, is loaded into a well formed in an agarose gel matrix. To prevent the DNA from diffusing out of the wells of the gel, a small amount of loading buffer containing glycerol is added to the DNA solution. The loading buffer also contains dyes that allow you to more easily track the progression of migration of the DNA. The agarose gel forms a mesh-like matrix through which the DNA can pass when a current is applied. DNA has a negative charge and will move away from the negatively charged terminal and towards the positively charged terminal at the distal end of the gel. Larger molecules of DNA will not be able to pass through the small gaps in the gel matrix as easily and quickly as the smaller sized DNA molecules can. The density of the agarose gel matrix determines the amount of separation between DNA polymers of different lengths. Since molecules of the same size move at an equivalent rate, a banding pattern will be observed, as represented in Figure 1. The dye(s) in the loading buffer run into the agarose gel along with the DNA, allowing you to estimate how far into the gel the DNA sample has run.
When a mix of DNA molecules is produced (e.g. by restriction enzyme digestion of a plasmid), it sometimes is necessary to purify a specific product (e.g. insert from a vector) from a gel to use in further manipulations. Provided the product is a specific size different from other products in the mixture, one can excise the desired band of DNA and extract it from the agarose gel.
The DNA molecules themselves will not be visible in the agarose gel, so it will be necessary to stain the gel/DNA with a dye. The most common dyes are intercalating fluorescent dyes that bind to double-stranded DNA in a sequence-independent manner. Since these molecules readily bind to DNA, they have been classified as mutagens/carcinogens (e.g. ethidium bromide). However, there are currently many safer dies on the market such as SYBR gold . Once bound to DNA, the fluorescence of the dye is increased dramatically, allowing one to visualize the DNA bands upon appropriate illumination of the gel (usually UV, λ = 300-365 nm).
Figure 1: The results of colony PCRs for 24 PCA Topo TA clones run on an agarose gel. The inserts for this set of blocks are ~620 bp. If you add the segment of vector backbone between insert ends and the M13 primers, the product size should be ~700 bp. Those clones missing a product of this size will not be processed further.
Agarose Gel Electrophoresis Materials
- Agarose Gel electrophoresis apparatus/chamber and gel tray; small agarose gel electrophoresis tank or large agarose gel electrophoresis tank large agarose gel electrophoresis tank
- well combs (6 or 10 for small tray; 15, 20 or 26 for large tray; 26 for 96-well plates)
- Bio-Rad Casting Stand Bio-Rad Casting Stand
- PowerPac Basic
- 250 mL Erlenmeyer flask
- 0.75 g UltraPure Agarose (Invitrogen, Product # 16500100)
- 1x TAE (48.4 g Tris base , 11.42 mL acetic acid , 20 mL 0.5M EDTA free acid pH 8.0; adjust final volume to 10 L)
- DNA gel loading buffer DNA loading buffer 6x
- DNA sample
- 1 kb DNA ladder 0.25-10 kb or 100 bp DNA ladder
- 1-20 uL pipette tips or 1-10 uL Rainin micropipette tips for multichannel micropipettor
- P20 micropipettor or P10 Rainin multi-channel micropipettor
- ChannelMate Reservoir System, 25 ml
- Insulated gloves/hot mitts
- UV transilluminator
- Light-proof tupperware staining container
- Mini blot mixer
- SYBR gold
Agarose Gel Electrophoresis Method
- For standard 1% agarose gels, measure out 0.4 g (small gel) or 0.75 g (large gel) of agarose and add to a clean 250 ml Erlenmeyer flask (hanging on rack over NW sink).
- Using a 50 mL graduated cylinder (hanging on rack over NW sink), add 40 mL (small gel) or 75 mL (large gel) 1x TAE buffer. Rinse the cylinder out with tap water and hang above NW sink to dry.
- Place the flask containing the agarose/TAE buffer in the microwave, and set the timer for 1.5 minute. It is important to monitor the flask as boil-overs are possible.
- After heating the agarose mixture, remove from the microwave and gently swirl the flask to mix the components (if you mix too vigorously when the flask is right out of the microwave, the solution will likely boil over onto your hand). If, after 2 min, there are still many particles of undissolved agarose floating in the solution, heat again briefly, and continue this process until there are no particles or swirls visible in the liquid upon mixing. Always mix by gently swirling to avoid introducing bubbles into the solution.
- Allow the flask to cool slightly ~15 min.
- In the meantime, for small gels, lubricate the tray gasket with a small amount of glycerol from the 15 mL tube in the DNA electrophoresis drawer. Ensure the gaskets are properly set and centered into the tray grooves, and push the tray into the gel chamber with the open ends and gaskets sealed against the chamber sides. Place desired comb(s) into the tray.
- OR In the meantime, for large gels, set up a gel casting stand with tray and seal the ends of the tray in the stand (the handle has a cog end that, when turned, will seal the ends of the tray). Level the trays using a bubble and the leveling screws built into the casting stands. Place desired comb(s) into the tray.
- Slowly pour the liquid agarose gel into one corner of the tray, being careful not to introduce bubbles. Use a pipette tip to remove any bubbles that may have formed. Rinse the flask with hot tap water and hang above the NW sink to dry.
- Allow the gel to completely set at room temperature, approximately 30 minutes.
- While the agarose gel solidifies, remove the DNA ladder from the freezer and keep in a microfuge rack to allow it to thaw.
- Carefully remove the combs, lifting first at one end to allow air to get into the wells. Wipe any liquid/gel off the comb(s) with a kimwipe and replace in the DNA electrophoresis drawer, along with the casting stand if using large gels.
- Reposition or Place the agarose gel into the gel running chamber, ensuring that the black cathode (-) is at top of chamber (at the back of the bench) and red anode (+) at bottom of the chamber (at the front of the bench) and that the wells are at the top of the chamber. DNA is negatively charged and migrates toward the positive red anode, moving from the top of the gel to the bottom.
- Fill the chamber with enough 1x TAE running buffer to barely submerge the agarose gel.
- Load the first and last wells (if there is room) of each row of wells with 5 µL of the DNA ladder. The ladder already contains loading buffer (it is blue). Use a fresh tip each time you pipette the ladder to prevent nuclease contamination from the TAE buffer.
- The method for dispensing samples will depend on the type of sample:
a) For samples that won’t be used in subsequent reactions (e.g. test PCR reactions or test RE digests), pipette 5 uL of gel loading buffer directly into the sample, mix carefully but thoroughly by trituration, ensuring not to generate bubbles, and remove 5 uL to load in a well. For 96-well plates, pour about 10 mL of Gel Loading Buffer (250 mL bottle in fridge) into a 25 mL disposable reservoir. Remove the sealing film from the PCR plate, being careful not to aerosolize droplets sue to vibrations. Use a P10 multichannel pipettor to draw up gel loading buffer. Mix with the samples directly in the plate, and dispense 12 samples at a time into 26-well gels. The spacing of the wells requires that the first row of the plate is loaded into alternating wells, and the next row of the plate is loaded into the remaining intervening wells. The first sample should be loaded into the second well of the agarose gel immediately after the ladder. Before dispensing, ensure that both the first and last tips are aligned with wells. Be sure to have the end of the tip IN the well, but not so deep that the bottom of the well is punctured – the DNA solution will sink to the bottom of the well due to the loading buffer density. Slowly remove the tip from the well, and eject the tip into the appropriate waste container.
b) For samples that will be used in further reactions (e.g. yeast cassette PCRs or RE digests for fragment purification), pipette a bead of 3 uL of gel loading buffer for each sample onto a strip of parafilm. Pipette up 5 uL (PCRs) or the full amount (RE digests) of sample and mix by trituration with a bead of gel loading buffer on the parafilm, maintaining the same order as the sample tubes. Dispense each mixed sample into a well of the agarose gel.
- Cover the apparatus with the lid to connect the electrodes in the appropriate orientation (black cathode (-) at top of gel and red anode (+) at bottom of gel).
- Turn on the power source and set it to run at 100V for 30 minutes (two rows of wells in large gels) OR until the dye front migrates the length of the agarose gel (or up to the next row of wells).
- Monitor the progress. When the current is first applied, take note of the abundance of bubbles along each strip of wire. More bubbles should be emanating from the top (cathode) strip of platinum wire because this is where hydrogen gas is accumulating from water hydrolysis, while the bottom (anode) wire is accumulating oxygen gas.
- Once the run is complete, turn off the power and transfer the agarose gel to the staining container. Thoroughly rinse the gel chamber and tray with tap water and hang above sink or place on drying rack (SW sink) to dry.
- Submerge the agarose gel in the SYBR Gold solution (50 μL of SYBR Gold stock diluted in 500 mL of deionized water), and set on the rocker for 20 minutes.
- Pour off the stain back into the SYBR Gold storage container while securing the agarose gel with your hand against the staining container to prevent the gel from falling out.
- Wash the agarose gel briefly in deionized water and leave some water in the staining container to aid in removing the gel for analysis.
- Place the agarose gel on the UV transilluminator in the gel doc system (room 168).
- Turn on the camera, gel doc unit and transilluminator. Set the gel doc unit to switch on the overhead white light (left knob), select the SYBR Gold filter (middle knob), and control the transilluminator with the safety switch (right knob).
- Log into the computer using your engineering login and open LabWorks 4.0.
- Close the pop-up window and click on the camera icon to open up a preview window. Using the slider at the top right of the window, set the preview exposure time to <1 s and center the agarose gel(s) using the preview image. Stop the preview and turn off the overhead light.
- Click the capture button. Adjust the capture exposure time as needed to increase the signal-to-noise of the image. Save the image as a tiff file to a USB flash drive or on your personal university server drive.
- Compare the bands to the nearest band in the DNA ladder. Cross-reference with the product insert to confirm the size of the band.
- Document your findings, and dispose of the agarose gel in the garbage. Replace the chamber and tray in the DNA electrophoresis drawer.
The DNA in agarose gels is traditionally stained with ethidium bromide. However, ethidium bromide is a potent mutagen and toxin. Much safer alternatives are available, such as the SYBR Gold used in this exercise. Although these alternatives are more expensive than ethidium bromide, the buffers and gels containing them do not have to be disposed of as hazardous waste.
Gel boxes vary in dimensions considerably depending on the manufacturer and model. Actual volumes required should be determined empirically or according to manufacturer instructions. The volumes for the agarose gel should be sufficient to produce a gel slab that is ~0.5 – 1 cm thick reaching high enough on the comb to give enough depth to the wells to load at least 20 µL of sample. A very thick gel will be more robust, but will also take longer to stain and the DNA bands will be fainter.
If the concentration of DNA in the sample is too low, the band may be very faint or not visible. In this case, re-run the gel with a larger volume of the DNA sample.
If the bands are not visible, and the ladder is more spread out than expected, it is likely that the gel was run too long, and the samples have run off of the gel.
Incomplete dissolving of the agarose in the buffer will result in wavy bands.
If the bands appear smeared, it is likely that the DNA has been degraded (if the smears are lower than the expected band size), or too much DNA has been loaded onto the gel (if the smears are higher than the band). Sometimes the bands across the entire gel will exhibit a “smiling” or “frowning” pattern. This is caused by excess voltage. Re-run the gel at a lower voltage for a longer run time.