Landscape of Interacting Microbes - PCR Protocols
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Making 20X SB Buffer for gel electrophorsis
| Chemical |
Amount |
| Sodium hydroxide (NaOH) pellets |
8 g |
| Boric acid (MW 61.83) |
47 g |
| Distilled water |
900 mL |
- Add both powders to 900 mL distilled water in a flask
- Stir with a magnetic stirrer and heat until dissolved
- Adjust pH to 8.0–8.2 with NaOH if needed
- Make up to 1 L with distilled water
- Filter sterilize using a 0.2 uM filter and store at room temperature
Dilute 20X SB Buffer to 1X SB Buffer (working solution)
| Solution |
Volume |
| 20X SB buffer |
50 mL |
| Distilled water |
950 mL |
Agarose gel preparation in SB buffer
- Add appropriate amount of agarose powder into 1X SB buffer in a flask, for example:
| % w/v |
Agarose |
1X SB volume |
| 1% |
1 g |
100 mL |
| 1.5% |
1.5 g |
100 mL |
| 2% |
2 g |
100 mL |
- Heat flask in a microwave (5–10 sec at a time) until dissolved, swirling to remove bubbles.
- Do not let the solution boil over!!! You will have to clean up the mess if the solution boils over!
- Add 10 µL SYBR Safe, ethidium bromide (or equivalent stain) per 100 mL to the flask.
- Insert combs into a gel tray
- Let the solution cool to ~30–40°C (until you can hold the flask with your hands) before pouring to avoid warping the gel tray.
- Pour into a gel tray with combs
- Move or remove bubbles with a pipette tip
- Allow the gel to solidify for at least 30 minutes
Gel electrophoresis
- Remove combs
- Load 7-10 uL of ladder into the first well
- Add 6X loading dye to PCR products at a 1:5 ratio
- Load 7-10 uL of PCR products with loading dye load into the remaining wells
- Load 7-10 uL of ladder into last well, if available
- Cover the gel tray and connect the cables to the power supply
- Run gel at 170V for 40 minutes (for quick resolution) or 135V for 65 minutes (for less smeared bands)
- Image using GelDoc