Agarose Gel Electrophoresis
This protocol describes the process of using Agarose gel electrophoresis to determine the size of PCR products as well as to distinguish polymorphisms between different genotypic variants of a PCR product.
B. Materials & Reagents
- a. Erlenmeyer flask, at least twice the volume of Agarose gel solution needed
- b. Graduated cylinder
- c. Pipettors and tips
- d. Weigh boat and spatula
- e. Scale, sensitive to 0.1 g
- f. Microwave
- g. Gel casting box, tray and combs (for forming shape of gel)
- h. Power supply device, for performing electrophoresis with gel
- i. Gel rig, for holding electrophoresis buffer and gel
- j. Cardboard box(es) or other chamber for keeping gel out of the light
- a. Agarose, low electroendosmosis grade
- b. 2.5x TBE buffer (Tris, Boric Acid, EDTA buffer)
- i. For 5L of 2.5x TBE buffer, combine:
- 1. 135 g Tris
- 2. 11.5 g EDTA
- 3. 69 g Boric Acid
- 4. Bring volume up to 5 L with ddH2O
- 5. Mix for 30 min at room temperature
- ii. Dilute this solution 1:4 for 0.5x TBE buffer to use in Agarose gel solution and in Gel rig
- c. SYBRSafe, 10,000x in DMSO
- d. ddH2O
- e. PCR product
- f. 3X STR
- g. DNA standard or ladder, with a range suitable for the expected PCR product(s)
1. For a medium size 1% gel (50ml, that holds two 17-well or two 24-well combs):
- a. Mix the following in an Erlenmeyer flask:
- i. 0.5 g Agarose
- ii. 25 mL 0.5x TBE buffer
- iii. Microwave this solution for 60 seconds, make sure the agrose is completely dissolved,then add another 25ml 0.5x TBE and 2.5ul of SyberSafe
- a. Place a gel casting tray into a gel casting box.
- b. Pour the gel solution into the gel casting tray and insert the comb(s).
- c. Make sure the gel solution surrounds each comb and is level. This is more of an issue with thicker (higher %) gels.
- d. Cover the warm gel with a cardboard box (so it will stay dark and protect the SYBR SAFE).
2. For thicker gels (higher %), increase the Agarose added to the TBE buffer in the first step:
- a. i.e. for one 50 mL, 2%, gel, use 1 g of Agarose. Mark volume on flask with marker. Always use large flask
- b. Microwave the gel for 2 minutes, swirling every 30 seconds. Make sure gel solution is thoroughly homogenized, i.e. clear and free of clumps and specks. If it is not, the gel picture may be difficult or impossible to read.
- c. If microwave time exceeds 2 minutes, the gel solution volume may drop noticeably below the marked line on the flask. Add ddH2O (~10% of total volume) to return volume to original level. This preserves the gel’s original concentration.
3. For multiple gels (up to 8 because we have 8 power supplies), increase microwave time, up to 3 or 4 minutes, following the above steps B(b) and B(c).
4. Let the gel sit until it is solid (about 10-20 minutes). Remove the comb(s) from the gel. Remove the casting tray (with gel still in this tray) from the casting box.
5. Put the gel (while it still rests in the gel tray) into a gel rig (electrophoresis apparatus) containing 250-275 mL 0.5X TBE. Always use the same buffer concentration you used to make the gel. Make sure the gel is just barely submerged in the buffer.
6. Load your samples and DNA ladder/standard(s) into the wells.
- a. 15 μL samples are first usually mixed with 3 μL 3X STR, unless PCR was performed with dyed reaction mix (i.e. GoTaq), or unless the PCR product is intended for sequencing, in which case aliquot 5 μL of the PCR product into a separate disposable plate and add 2 μL 3X STR to that for the electrophoresis process. 3X STR interferes with sequencing.
- b. Choose a DNA ladder/standard that will show bands smaller and larger than the anticipated size of your product, i.e. a 100 bp ladder (which shows bands all the way up to 2 kbp) would be appropriate for any product in between 100 bp and 2 kpb.
7. Place the clear gel rig lid securely onto the gel rig, and plug in the power supply.
8. Turn on the power supply, press the voltage button to select your desired voltage (low voltage is better for thicker gels and smaller polymorphisms).
9. Press “Set” on the power supply, and then an arrow key to set the desired run time in minutes. Then press “Set” again to start. For simply checking if a PCR has worked, running a gel at 125V for ten minutes should be fine.
10. Take photograph, using the UV-transillumination setting on the Gel Logic 200 imaging device, and the Kodak software. An exposure time of 2 seconds should be sufficient for a normal gel. Save your picture.
11. Dispose of gel in the trash, or run it longer in the gel rig as needed.