If you are using DNA or doing PCR’s in your research, then you can guarantee you need to make up agarose gels at some point. I have lost count of how many agarose gels I have made during my time in the labs. Here is my guide on preparing and making agarose gels:
1. Decide what percentage gel to make
You should adjust the concentration of the gel per the size product you are expected to see. As a rule of thumb, low percentage (0.8 – 1%) gels should be preferred when you want to separate very large molecular weight DNA, such as genomic DNA. Higher percentage gels (1 – 3%) are chosen when you want better resolution, usually to visualise smaller bands (< 500bp), such as those generated in real time PCR.
2. Calculate the amount of agarose powder and volume of TBE buffer
It is best practise to do all your calculations before you start. You need to decide what amount of agarose powder and the volume of tris/borate/EDTA (TBE) buffer to combine to make your required percentage gel. The volume of TBE will depend on the gel casting tray size you are using. An easy way to work this out is to add water to the tray until it is full, and then measure the volume of water used via a measuring cylinder.
Here are some examples of agarose gel solutions to make when using a 50 mL gel casting tray:
- 1% gel = 50 mL 1x TBE buffer and 0.5 g agarose powder
- 2% gel = 50 mL 1x TBE buffer and 1.0 g agarose powder
- 3% gel = 50 mL 1x TBE buffer and 1.5 g agarose powder
To work out the amount of agarose powder required, when you know the volume of TBE buffer and the percentage gel desired, you can use the following equation:
Agarose (grams) = Gel desired (%) x Volume 1x TBE buffer (mL)
3. Preparation of TBE buffer (if required)
First off you will need some TBE buffer. Some of you folk may be able to purchase ready-made TBE buffer, which is totally fine, but it is so simple and cheap to make. It is best to make a 10x stock solution and then use this to make a 1x working solution. Use our recipe for 10x TBE buffer here.
Making the agarose gel
Now you have your TBE working solution, you are ready to make the agarose gel:
- Start by weighing out the amount of agarose powder you require and transfer this to a conical flask. Whilst heating the mixture to dissolve the agarose, the solution will bubble up so make sure you select a conical flask that is large enough to hold the mixture and has room to account for the bubbling solution.
- Measure out the volume of TBE working solution required for the agarose gel. Add this to the conical flask containing the agarose powder. Give the mixture a swirl.
- Heat the solution by putting the flask in a microwave. Make sure you keep an eye on the heating solution. If you over heat, you will find your solution all over the microwave. It is best to heat little and often by opening the microwave frequently to inspect the solution.
- Once all the powder has dissolved, remove the flask from the microwave and place on the bench to cool. Be careful, as the flask will be boiling hot!
- When the solution is cool enough that you can handle it with a gloved hand comfortably, then you are good to add your nucleic acid staining solution of choice.
- In our lab, we use Midori Green Advance (Nippon Genetics), a non-carcinogenic dye. For this dye, you need to add 0.5 μL of Midori Green Advance solution for every 10 mL of agarose gel solution. Swirl the flask to mix the dye. Make sure all the dye is mixed into the solution completely.
- Pour the solution into a gel cast tray containing the gel combs. Don’t make the gel too thick. About half way up the combs should be enough. Also, try not to introduce any bubbles. If you do see bubbles, especially around the gel comb, get a pipette tip and pop it. Or move it to the bottom of the gel. If you have any agarose gel mixture left over, pour this into an unused casting tray to set. Do not pour it down the drain.
- Leave the gel mixture to set. The time required for the gel to completely set with vary depending upon the gel concentration and the surrounding temperature. Higher percentage gels set faster than lower percentage gels and cooler environments also set gels faster than warmer ones. I usually leave mine to set for at least 15 minutes.
- Carefully remove the combs without splitting the gel, especially around the wells.
- Transfer the gel into the gel electrophoresis tank and fill up the tank with 1x TBE buffer. Depending upon the tank size this may require a considerable amount of working TBE buffer. Make sure the solution fully submerges the agarose gel. Inspect that there are no air bubbles in the wells. If so, use a pipette tip to pop it out. Also, make sure the anode (positively charged end, which is red in colour) is at the bottom end, so that the gel will run from negative (negatively charged end, which is black in colour) to positive.
- The gel is now ready to be loaded with samples.
Preparing samples for loading
Before you load the samples onto the gel, you will need to mix it with a DNA gel loading dye. The loading dye is required for two things: firstly, to make your samples more visible during the loading and running of the gel; secondly, to enable the samples to sit and stay in the agarose wells.
You can usually get DNA gel loading dye when you purchase DNA ladders. They should state their concentration on the tube, such as 5x. So, depending upon the total volume you want to load in each well (usually between 5 – 20 μL) you need to work out how much loading dye to mix with your sample. It is best to do this is PCR tubes.
If you want to load 10 μL into each well and you have a 5x loading buffer, then mix 8 μL of sample with 2 μL of loading buffer in a PCR tube.
Once your samples are mixed with the loading dye, then they are good to be loaded into the wells.
Loading and running the agarose gel
Load all the samples into the appropriate wells. Take care when pipetting to avoid stabbing the gel. It is best practise to pipette the samples out slowly to prevent them from coming back out of the wells.
Also, add a DNA ladder so you have a size reference when looking at your results. Select a DNA ladder with a large range of sizes which covers the size you are expecting in your samples.
Finally, add the lid onto the tank and connect it up to a power pack. Choose an appropriate voltage (V). A high voltage (e.g. 100 V) will migrate the samples through the gel faster than a slow voltage (e.g. 40 V). Don’t run the samples on too high a voltage (> 100V) as this can heat up the TBE buffer and cause the agarose gel to melt slightly. You also need to think about the size of the gel. Obviously, larger gels can run for much longer than shorter ones before your samples run off the end. Also, when using higher percentage gels, you will notice the migration of your samples is much slower compared to lower percentage gels.
I usually set my power pack to 60 V for about 60 – 90 mins. Whatever voltage you decide, be sure to keep on checking the gel to make sure you can still see the samples. Once the samples are near the bottom of the gel, you are good to stop the electrophoresis and inspect your gel using a UV transilluminator.
Featured image credit: PlaxcoLab (via Flickr)