UltraPure™ Agarose is a standard melting temperature, multi-purpose agarose that is ideal for routine separation analysis. UltraPure™ Agarose resolves DNA and RNA fragments from 500–23,000 bp, and has no detectable DNase or RNase activity. It can also be used for:
- Analytical separation of DNA, RNA, and PCR fragments
- Recovery of DNA, RNA, and PCR fragments
- Southern and northern blotting of fragments
- Ouchterlony and radial immunodiffusion (RID)
Specifications 1.5% Conc.
Gel Strength* ≥ 1,200 g/cm2
Gel Point ≤ 36°C
Melting Point ≥ 90°C
* Gel strength is calculated at 1% concentration.
Usage
Refer to the table below for the recommended concentration of UltraPure™ Agarose needed to resolve DNA fragments of the approximate listed range:
Fragment Size | % Agarose (in 1X TAE) | % Agarose (in 1X TBE) |
1,000–20,000 | 0.60 | 0.50 |
800–10,000 | 0.80 | 0.70 |
400–8,000 | 1.00 | 0.85 |
300–7,000 | 1.20 | 1.00 |
200–4,000 | 1.50 | 1.25 |
100–3,000 | 2.00 | 1.75 |
Method 1: Microwave- Determine the amount of agarose solution needed to cast your gel.
Note: Remember to take the thickness of the gel into account, as it affects both well volume and power requirements.
- Add room temperature buffer (TAE or TBE) into a flask that can hold 2–4 times the volume of your agarose solution. Place a magnetic stir bar into the flask.
- Put the flask on a magnetic stirrer and slowly sprinkle the required amount of agarose powder into the flask as the solution mixes, to prevent the formation of agarose clumps.
- Remove the stir bar.
- Weigh the flask and solution before heating.
- Cover the mouth of the flask with plastic wrap, and pierce the wrap with a small hole for ventilation.
- Place the flask in the microwave oven and heat the solution until bubbles appear.
- Remove the flask carefully, and swirl gently to resuspend any agarose particles. Exercise caution – microwaved solution may become superheated and foam over when agitated.
- Reheat the solution until the solution comes to a boil, and all the agarose particles are dissolved.
- Remove the flask carefully and swirl gently to mix the solution.
- Place the flask on a scale, and bring it back to its initial weight (from Step 5) with warm distilled water.
- Mix gently and cool to 50–60°C (at room temperature for at least 20 minutes) before pouring the solution into the casting tray.
Method 2: Boiling water bath
- Determine the amount of agarose solution needed to cast your gel. Note: Remember to take the thickness of the gel into account, as it affects both well volume and power requirements.
- Add room temperature buffer (TAE or TBE) into a flask that can hold 2–4 times the volume of your agarose solution. Place a magnetic stir bar into the flask.
- Put the flask on a magnetic stirrer and slowly sprinkle the required amount of agarose powder into the flask as the solution mixes, to prevent the formation of agarose clumps.
- Weigh the flask and solution before heating.
- Cover the mouth of the flask with plastic wrap, and pierce the wrap with a small hole for ventilation.
- Bring the solution to a boil while stirring, and allow it to boil gently for approximately 10 minutes or until the agarose is completely dissolved.
- Place the flask on a scale, and bring it back to its initial weight (from Step 4) with warm distilled water.
- Mix gently and cool to 50-60°C (at room temperature for at least 20 minutes) before pouring the solution into the casting tray.
Visualization of DNA
For visualization of DNA in the gel, a fluorescent dye can be added to the agarose solution just prior to pouring, or the gel can be stained after electrophoresis. For the intercalating dye ethidium bromide, use a final concentration of 0.5 μg/ml. If more sensitive detection is required, use SYBR® Green I nucleic acid gel stain (Invitrogen Cat. no. S-7563), or SYBR® Safe DNA gel stain (Invitrogen Cat. no. S33102). Use of SYBR® Safe DNA gel stain in conjunction with blue light transillumination is recommended for gel extraction procedures for cloning purposes. Refer to the appropriate instructions for these products for in-gel staining, or post-staining protocols.
Dye Mobility
Refer to the following table for the migration of Bromophenol Blue and Xylene Cyanol tracking dyes in relation to DNA:
% Agarose
| Bromophenol Blue |
| Xylene Cyanol |
|
| TAE
| TBE
| TAE | TBE
|
0.3 | 2,900
| 2,850
| 24,800 | 19,400
|
0. 5 | 1,650
| 1,350
| 11,000 | 12,000
|
0.75 | 1,000
| 720
| 10,200 | 9,200
|
1
| 500
| 400
| 6,100
| 4,100
|
1.25
| 370
| 260
| 3,560
| 2,500
|
1.5
| 300
| 200
| 2,800
| 1,800
|
1.75
| 200
| 110
| 1,800
| 1,100
|
2
| 150
| 70
| 1,300
| 850
|