Intro: How to Prepare an Electrophoresis Argarose Gel
This instrucable illustrates the process of casting, loading, and processing an electrophoresis argarose gel. Gel electrophoresis separates biological molecules based on size and weight by utilizing electricity. Polar molecules move through the gel at different rates resulting in distinct bands. The location of these bands, relative to a standard, indicates the approximate size of the material in each sample. These samples usually consist of DNA, RNA, or protein molecules. The uses of gel electrophoresis include: estimation of the size of cloned DNA, analysis of PCR products, or separation of genomic DNA. Because chemicals used in gel electrophoresis can be hazardous, no one should attempt casting a gel without basic lab safety training. Also, anyone attempting to photograph the gel for analysis should be trained on using a UV camera. After adequately preparing stock solutions, casting a gel can take up to an hour depending on the number of samples. Don't worry though. Most of the time is spent waiting for the gel to try and process and the gel. After reading this instructable, you should not have problems performing argarose gel electrophoresis, which is a common process utilized in many biological labs.
Step 1: Gather Materials
Stock Solution Chemicals
1) 50X TAE solution
2) 1 liter plastic bottle
3) 250 milliliter flask
4) 2.5 g agarose
5) Distilled water
1) Gel plate
2) Gel form
1) Pipette aid
2) Pipette tips
4) Samples with dye
5) Ethidium Bromide
6) Plastic pipette
7) Gel box
8) Gel box lid
8) Power supply
9) UV camera
2) Lab coat
3) Safety glasses
4) Oven mitts
NOTE : Before starting any of the procedure, make sure to wear gloves, a lab coat, and eye protection. See http://fscimage.fishersci.com/msds/45442.htm for MSDS on Ethidium Bromide.
WARNING : Ethidium Bromide is a known mutagen as stated in the MSDS. Handle with care as specified by your laboratory procedures.
Step 2: Prepare Stock Solutions
Preparing stock solutions allows you to caste multiple gels over a short period of time. This saves time and is more efficient. Store the stock solutions in the gel electrophoresis area in your lab. Make sure to label each stock solution appropriately. If you already have these prepared in your lab go to the next step.
1) Prepare 1X TAE Stock Solution
a. Pour 20 ml of 50X TAE solution into 1 liter plastic bottle
b. Bring final volume to 1 liter with distilled water
c. Gently shake solution
NOTE : Many labs have 50X TAE solution on hand. If your lab doesn't, click on this link to find instructions on making one.
2) Prepare 1X TAE + 1% Argarose
a. Add 250 ml of 1X TAE solution in 250 ml flask
b. Add 2.5 g of Agarose
c. Boil in a microwave
WARNING: Solution will be very hot! Handle with care and use oven mitts.
After stock solutions are prepared, the next step is to cast the gel.
Step 3: Cast the Gel
The argarose gel acts as a medium for the molecules to pass through during electrophoresis. Wells, created by the comb, contain your samples during the electrophoresis process. At room temperature, the stock solution 1X TAE + 1% argarose gel is a solid. When casting the gel, the solution must be a liquid to form into the plate mold.
1) Place gel plate inside plate form
TIP: Although the gel form supposedly protects against leaks, it is not full proof. When attempting to caste a gel for the first couple of times, try to tape the edges as shown in the second picture.
2) Turn front dial to tighten into place
3) Attach comb to plate
4) Boil 1X TAE + 1% Argarose solution in microwave
This should take around 5 minutes
WARNING: Solution will be very hot! Handle with care and use oven mitts.
5) Pour hot solution into gel plate
For best results, make sure solution is completely liquid before pouring into plate. Supply enough solution to adequately create wells on the plate.
6) Wait for solution to solidify
This should take around 5 to 10 minutes.
The mold should be solid before proceeding to load your samples.
Step 4: Load Samples to Gel
Loading the gel can be tricky and time consuming depending on how many samples you have. When loading samples to the gel, find a way to stabilize your hand to avoid puncturing your gel. Your samples should contain approximately 2% dye.
1) Loosen dial on the plate form
Remember to remove the tape if you used any.
2) Slowly and carefully remove the comb.
CAUTION: Avoid puncturing or damaging the gel.
3) Place plate with gel into gel box as shown.
4) Slowly pour 1X TAE stock solution into gel box
5) Add enough solution to fill the chambers and supply a thin layer of solution above the gel
If 1X TAE level does not adequately cover the wells, add more to gel box.
6) Pipette 10 µl of ladder into the leftmost well as shown in the third picture.
CAUTION: Do not puncture the bottom of the well with the pipette tip. For best results, insert the tip barely inside of the well.
7) Pipette 10 µl of samples and dye into the following well using the same technique
TIP: Record the placement of each sample to the corresponding well. This will make things much less confusing when taking pictures of the gel.
The next step is to process the gel using electrophoresis.
Step 5: Process Gel
Gel electrophoresis separates biological molecules based on size and weight by utilizing electricity. Polar molecules, such as DNA, move through the gel at different rates resulting in distinct bands. The longer the plate is exposed to electricity, the more distinct the bands become.
1) Using a plastic pipette, add several droplets of ethidium bromide to the front, middle, and back chambers of the gel box
WARNING: Ethidium Bromide is a known mutagen. Handle product with extreme care. Remove gloves after dealing with Ethidium Bromide and dispose of properly before proceeding to the next step.
2) Attach the lid to gel box.
Make sure to match up black electrodes with red electrodes.
3) Plug cords into power supply
4) Set desired voltage on monitor
This depends on your gel, but a safe voltage to use is 90V.
5) Push the run button and let electrophoresis run for 20-30 minutes
TIP: You do not need to watch the gel during this entire process. However, wait until small bubbles form on each end before leaving the area. Periodically check to see if orange bands are moving down the gel from the wells as shown in the fourth picture.
6) Turn off power supply
The gel is now ready to be photographed by a UV Camera.
Step 6: Photograph Gel Using UV Camera
Ethidium bromide is often used as in indicator because it binds with DNA. When exposed to ultraviolet light, it will fluoresce with an orange color showing the distinct bands of each sample. This can then be compared to the ladder.
1) Unplug cords from power supply
2) Remove lid from gel box
3) Carefully remove plate and gel
TIP: To remove excess liquid between the plate and gel, use a paper towel.
4) Place gel and plate onto a UV camera
5) Take a picture of the gel using the UV camera
WARNING: Do not look directly into UV camera surface when on as it can be damaging to eyes.
After completing gel electrophoresis, taking a picture of the gel allows you to analyze the results of the procedure.The second picture illustrates an example of a photographed gel. The ladder is show on the left side. Distinct bands are illuminated in the picture. Wells are labeled to keep everything organized. Comparing the sample bands to the standard allows the user to estimate the size of each sample. Electrophoresis allows you to analyze many different things, such as PCR product or DNA size. Knowing how to perform this common lab procedure is key to being productive in a biological lab.