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- Microarraying FAQS
- Electrophoresis FAQS
1. Microarrying FAQS
Agarose (Horizontal) Gels
Q:What are micro-titer format combs?
A:The teeth of Galileo micro-titer combs are spaced to align with 8 & 12 channel pipettes. 1X micro-titer combs are spaced 9mm apart on centre and will align perfectly with multichannel pipettes. 2X micro-titer combs have teeth spaced 4.5mm apart, so that the pipette will fill every other well. Then simply by moving the pipette 1 well over (½ pipette space) the other set of wells can be filled. This sequence is repeated until all wells are filled.
Q: How can a shorter gel be cast on large horizontal devices?
A: When a shorter gel length is desired (usually because you have fewer samples to run) a Galileo casting dam can be inserted in the gel tray to seal the tray at any desired length.
Q: What is the best way to load a gel?
A: The best way to load gels is to first make sure the fully solidified gel is submerged in the running buffer with only a thin layer of buffer over the top of the gel. While pipetting their samples into the gel, most researchers guide their pipettor with their other hand to prevent the tip from damaging the gel matrix. Since loading buffer is more dense than running buffer, the pipette tip can be held slightly above the well during filling, and the sample will flow down into the well. Be sure to slowly dispense the sample to avoid sample loss.
Q: How hot should my agarose be when I pour my gels?
A: For uniform gels, your agarose must be must be completely dissolved and free of any particles. Remember that agarose is a long-chain polymer and takes some time to fully dissolve into solution. A good way to check your gel material is to carefully swirl it while holding it up to the light to look for any “glass-like” particles that haven’t dissolved. Continue heating until the gel looks clear, like water. Remember to compensate for evaporation as continued heating will cause your gel% to increase. When the gel has been dissolved, cool it to approximately 60°C before pouring into the gel tray.
Q: How do I know if my gel box is running?
A: The easiest way to tell if your device is working is to look for the bubbles rising off the electrodes at each end of the device. The negative end (black) also called the cathode, should have approximately twice as many bubbles forming as at the (red) anode end. If no bubbles are forming, check that the power supply is on, in run mode, and that all connections are secure. If bubbles are forming, then current is running through the system. But remember however, that proper buffer concentration and age greatly affect the elctrophoresis process. If samples are not running properly, then the most likely problem is poor buffer formulation.
Q: What direction should my gel run?
A: The phosphate backbone of DNA gives the molecule a uniform negative charge. So it will move toward the positive anode (red) electrode. The easiest way to remember this is “run to red”.
Q: Why does my DNA run slower in one device versus another when all of the experimental parameters (buffer, % agarose, voltage, thickness of gel, etc.) are identical?
A: The critical factor when comparing two devices is the inter electrode distance. This is the distance (not the length of the gel) that determines field strength. (See question below) The power supply setting is based on this value. For example: to duplicate run conditions of 5volts/cm in two devices with 10 and 12 cm interelectrode distances, the voltage applied must be 50 and 60 volts respectively.
Q: How do I calculate volts/cm for each gel box?
A: Remember that since the electrical field generated in the device includes the length of the buffer as well as the gel, all volts/cm calculations should be based on the distance from one gel box electrode location to the other electrode, NOT on the length of the gel. The standard run value range is 3-6 volts/cm for most DNA gels.
Q: Can I run my DNA samples and stain the gel the following day?
A: This can be done as long as the gel remains under buffer or distilled water, however there can be some diffusion of the DNA in the gel, so visualizing your samples immediately will give clearer, sharper bands.
Polyacrylamide (Vertical) gels
Q: Please explain Notched vs Plain Glass?
A: When using vertical gel electrophoresis devices, spacers are sandwiched between one notched and one plain glass to form the gel “cassette”. The notched glass plate provides a contact area for the buffer from the upper buffer chamber to flow over the gel to complete the electrical circuit required for electrophoresis. Note that the “ears” on notched plates are fragile, and should be handled with care. Extra notched and plain glass plates are available for all Galileo vertical units.
Q: How can I avoid bubbles at the base of my vertical gels?
A: If there are voids at the base of a vertical gel, bubbles can be trapped when the gel cassette is placed in the running buffer. These bubbles will cause an uneven electrical field and poor gel performance. Galileo vertical gel systems feature a cast-in-place system that that forms a smooth, flush seal at the bottom of the gel cassette. This allows for full filling and polymerization (no oxygen exposure) along the bottom edge of the gel, preventing any voids which would trap air and cause bottom bubbles.