Agilent Technologies · Transcript · · Updated
SurePrint: SurePrint Microarray Washing – Transcript
Video by Agilent Technologies · Watch the original on YouTube
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- The speaker demonstrates the setup of staining dishes containing wash buffer one at room temperature to disassemble the microarray hybridization chamber.
- Microarray slides are submerged in wash buffer one and separated from the gasket slide using forceps.
- After completing wash one, the slide rack is transferred to wash buffer two while minimizing buffer carryover.
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So first, what I would like to do is go over our washing station that we have set up here. As you see, we have a variety of staining dishes, and each staining dish is a different buffer that we'll be using depending on the step of the wash procedure that you'll be running. In our first dish here, we have wash buffer one, which is simply at room temperature, and what we'll be using this staining dish for is to disassemble our microarray hybridization chamber.
Next on a stir plate, we have another staining dish that has wash buffer one in as well, and inside there's a stir bar as well as a slide rack that we'll be using to wash the array in wash buffer one. Again, this is at room temperature. Finally, we have an additional stir plate, and this particular staining dish is currently empty because what we suggest is you pre-warm wash buffer two to the appropriate temperature the night before you plan to use it, and then dispense it into the dish right before
you're ready to wash your samples in wash buffer two. Now for other applications such as array CGH or array CGH plus SNP, there's a need to have a much tighter control over the temperature, so to help accomplish this, we have a slightly different setup for wash buffer two, and that's to use a Pyrex dish that has water inside about a quarter to halfway filled up, and then an empty staining dish that we'll cover with aluminum foil, and also place it 37 degrees overnight to get to temperature, and what
this will allow is for us to have much tighter control over that 37 degree temperature point during that particular wash step. So once the hybridization is complete, you can remove the hybridization chamber from the oven and disassemble it at room temperature. Then what we'll be doing is taking out the micro array and gasket slide, submerging them in wash buffer one, and then disassembling the micro array from the gasket slide to start the washing process.
Prior to moving on, I like to visually inspect the arrays to ensure that the hybridization solution between the gasket slide and the micro array slide is at least halfway if not higher within the region that the array is contained in.
If you do see any evaporation in any of the particular arrays, simply just make a note of it in your log. So carefully unscrew the top, slide that off, remove the cover, they'll now be exposed the array and the gasket slide by grabbing the edges, carefully remove it from the hybridization
chamber, and submerge it underneath your first staining dish containing wash buffer one. Now using a pair of forceps, carefully wedge the forceps between the two slides and gently twist.
This will allow the gasket slide to fall to the bottom, and you'll be holding the array slide. Now while keeping the array slide completely submerged under the wash buffer, simply swirl it a couple of times to help remove any residual wash buffer or hybridization solution that's on the array, and then quickly transfer it to the first wash on the magnetic sand.
If you're going to be washing more than one glass slide at a time, make sure they're evenly distributed across the slide rack in the wash buffer one, and typically we recommend processing no more than five glass slides at a time at this step. Once all the glass slides have been loaded and evenly distributed, you can then turn on the stir bar to the medium to medium low setting and allow the wash to take place.
When wash one is almost complete, you can go to the 37 degree incubator and retrieve the wash buffer two that's been warming. Quickly pour the wash buffer two into the second staining dish on its own magnetic stir plate, and then go back over to the first staining dish and transfer the slide rack from the first container to the second container containing wash buffer two
while trying to minimize the amount of buffer carryover. Once the rack has been transferred, start the stir bar to a medium to medium low setting. Once the stir bar has started spinning, you can initiate wash two for the appropriate amount of time.
If you're going to be processing microarrays that require a much more stringent wash two step, such as array CGH or array CGH plus SNP, prior to the end of wash one, go to the incubator that has the glass dish as well as the staining dish that has been incubating overnight, and then promptly add the wash two that's also been warming at 37 degrees into the staining dish.
Once the wash has been added, go back over to the initial wash one container and transfer the slide rack while minimizing buffer carryover from the first dish into the second dish. Once the transfer is complete, you can activate the stir bar to a medium or medium or low setting.
If you have a stir plate that also has the ability to control temperature, you can also set your stir plate temperature to 37 degrees ahead of time to again reinforce the temperature control that's going to be occurring during wash two. Once wash buffer two is complete, very slowly remove the slide rack from wash buffer two as this will allow the microarrays that have been washing in the solution to essentially be dry upon removal.
If for some reason you see water droplets on the slides, simply submerge the slide rack back into wash buffer two and then slowly remove it again as you want to make sure that the slides will be dry upon removal from wash two.