Sonication protocol for the 454 PicoTiterPlate

version 1.0  5-20-06

Roe-Lab University of Oklahoma

 

Materials needed:

 

1.   VWR model 50T sonicator

2.   A plastic rod for lifting the PTP.

3.   A plastic PTP holder with an open bottom that will hold the PTP ~2 cm above the bottom of the sonicator tub but still let the waves through to hit the PTP.  We glued plastic legs onto the tan plastic top of an old ABI 96 well viper plate holder. The thick black plastic base is not used.

 

Note:  The PTP is made of glass. It will chip or break if it touches the metal tub while the sonicator is running. The sonicator element is mounted under the center of the bottom of the tub. The PTP holder keeps the PTP away from the very high intensity right next to the sonicator element.

 

Hold the PTP only by the edges and corners. Do not touch the center regions of the plate. After cleaning don't rub the front or back surface of the PTP. You may force lint into some of the wells on the front or scratch the back of the plate that the camera sees. Pat the PTP dry with Kimwipes.

 

1.   Place the PTP holder in the bottom of the sonicator. Place the PTP on the holder faceup. Fill the tub with dH2O up to the fill line. Let the PTP sit in the water for 2-3 minutes to partially hydrate the surface. (This dramatically shortens the sonication time to ~5 minutes.)

 

2.   Turn the sonicator on. Within a minute you will see the brown color lift from the PTP like a thin film and move across the PTP. This "film" of beads and bubbles dissipates into the water turning it a light brown color. To remove more of the beads, move the PTP from side to side with the plastic rod. Lift one side of the PTP up with the plastic rod, rotate the PTP 90 degrees, and lay it back down on the holder. Again move the PTP from side to side with the plastic rod.  At this point the center of the PTP should be almost clean. There may still be an brown outline around each region of the PTP where the bead-loading gasket was positioned.

 

3.   To clean the edges of the PTP, use the plastic rod to lift the PTP up onto one edge. Hold the top edge of the PTP by the corners (that are out of the water) vertically so that the bottom edge is touching the holder above the sonication element. You should see the brown color near the bottom edge gradually go away. Every 10 seconds or so move the PTP a little bit until all of the color along the bottom edge is gone.

 

4.   Rotate the PTP 90 degrees after approximately a minute so that the next edge is near the sonicator element and repeat step #3.  Rotate the PTP by 90 degrees two more times at ~1 minute intervals to clean the other two edges. The total sonication time is about 5 minutes.   There may be small brown dots or smudges left on the PTP where the input and output ports of the bead-loading block were when the PTP was loaded. The beads in these spots are hard to get rid of, but do not cause a problem. They are outside the view of the camera.

 

5.   Turn the sonicator off. Remove the PTP from the sonicator and rinse both sides well with running dH2O. Hold the PTP vertically and let the water run off the front surface for a while. Lay the PTP facedown on a large Kimwipe folded into three layers. Pat the back lightly with another Kipwipe to get rid of the standing water. Place the PTP on a dry Kimwipe faceup in a safe location. Lightly cover the PTP with a dry Kimwipe and let it air dry.

6.   Using this cleaning protocol we have successfully reused both half and full PTPs over 20 times, having not yet reached a limit for re-use.

 

  • View a Quicktime, time-lapse movie of a typical sonication cleanup

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    Bruce Roe, broe@ou.edu