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Pre-Cast Gels for Safe Nucleic Acid Analysis

互联网2013-11-13

1029

实验步骤

1. Loading E-Gel® with SYBR Safe™ Gels

1) Pre-running E-Gel® with SYBR Safe™

i. Plug the E-Gel® PowerBase™ v.4 into an electrical outlet using the adaptor plug.

ii. Open the package containing the gel and insert the gel (with the comb in place) into the apparatus right edge first. Press firmly at the top and bottom to seat the gel in the base. You should hear a snap when it is in place. The Invitrogen logo should be located at the bottom of the base, close to the positive pole. See the diagram below. A steady, red light will illuminate when the E-Gel® gel is correctly inserted (Ready Mode).

iii. Press and hold either button until the red light turns to a flashing green light. This indicates that the 2-minute pre-run has started.

iv. At the end of the pre-run, current will automatically shut off. The flashing green light will change to a flashing red light and the PowerBase™ will beep rapidly.

v. Press and release either button to stop the beeping (you will hear only one beep). The light will change from a flashing red light to a steady red light.

2) Method of Loading Samples

i. Remove the comb from the E-Gel® with SYBR Safe™ gel using both hands to lift the comb gently by rolling the comb slowly towards you. Be careful to pull the comb straight up from both sides. Do not bend the comb. Remove any excess fluid using a pipette.

ii. Load 20 µl of sample per sample well.

iii. Load 20 µl (500-700 ng) of the appropriate molecular weight markers.

iv. Load 20 µl of water into any remaining empty wells.

2. Running E-Gel® with SYBR Safe™ Gels

1) Choose the 30-minute run for E-Gel® with SYBR Safe™ gels. Press and release the 30-min button to start the electrophoresis run. The light will change to a steady green light.

Note: The actual running time may vary between 30-33 minutes.

2) Current through the E-Gel® gel automatically shuts off at the end of each run. The E-Gel® PowerBase™ v.4 signals the end of the run with a flashing red light and rapid beeping.

3) Press and release either button to stop the beeping. The light will turn to a steady red light.

4) At the end of the run, remove the gel cassette from the power unit and analyze your results as described in the next sections.

3. Visualizing E-Gel® with SYBR Safe™ Agarose Gels

While yielding similar sensitivities to ethidium bromide, SYBR Safe™ is somewhat dimmer yet with a lower background than ethidium bromide. As a result a slightly longer exposure time, or higher gain setting may be necessary.

4. Opening an E-Gel® with SYBR Safe™ Cassette

1) Place the E-Gel® Opener on a flat surface, with the knob extending off the edge of the laboratory bench and facing the user. Set the E-Gel® Opener to its widest open position by turning the knob counterclockwise.

2) Insert the E-Gel® into the E-Gel® Opener so that two opposing sides of the gel cassette are aligned with the blades (see Figure 1). Position the E-Gel® such that the two sides fit into the grooves housing the blades.

3) Turn the knob steadily clockwise to bring the blades in contact with the E-Gel® cassette. As the knob is tightened, you will hear a series of pops. Continue to turn the knob until the resistance increases. Stop turning the knob as soon as the E-Gel® cassette begins to lift off the surface of the platform. Two sides of the E-Gel®will now be unsealed. Note: Once you observe the E-Gel® cassette begins to lift off the surface of the platform, do not continue to tighten the knob, as you will damage the E-Gel®.

4) Unscrew the knob and remove the E-Gel®. The E-Gel® cassette fits snugly in the recessed groove, and you may have to carefully work the cassette from the housing. Turn the E-Gel® 90° and re-insert the gel cassette into the Opener so that the two remaining sealed sides can be opened.

5) Repeat Step 2 to open the remaining two sides of the E-Gel®. Stop turning the knob when you see the top of the E-Gel® cassette begins to lift off the gel.

6) Unscrew the knob and carefully remove the E-Gel® cassette. The 4 sides of the cassette should be unsealed. If not, repeat Steps 2-5 as necessary. Remove the E-Gel® and set the opened cassette on your bench.

7) If you plan to blot the gel, do not pick up the gel from the cassette. Lift off the top of the gel cassette. Place the blotting membrane on the gel and pick up the cassette with the gel and membrane. Flip the gel and membrane out of the cassette onto your gloved hand and then flip the gel and the membrane directly onto your wet blotting paper.

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