Array Hybridization and Washing

Materials Needed

Hybridization Post Hybridization Washing
Hybridization Chambers
Lifter Cover Slips (Dust Free)
100 C Heat Source
65 C Heat Source for Hyb Chambers
poly dA 10 mg/ml
20X SSC, 3X SSC
10% SDS
 3 Slide Wash Chambers
Slide Rack
20X SSC
10% SDS
Centrifuge that can spin plates at 500 RPM

Probe Preparation

Probe can be dried down to an appropriate volume for adding probe mix compoments. Add SDS last, just prior to denaturation to avoid preciptation on ice.
Use 10-12 uL probe mix per hybridization with regular cover slips,
use 20 uL probe mix per hybridization with lifter slips.

Probe Mix: 3X SSC
1 mg/ml poly dA (10 ug)
0.2% SDS 		notes
Don't worry about the in-accuracy
of adding sub-microliter amounts
of 10% SDS.

Add SDS to probe and Denature at 95 C, 2 min.

It's important to cool the probe down before pipetting it onto the array, however you can't place it on ice because the SDS will precipitate. Spin the probe briefly to pellet condensation. Some microfuges which allow the tube to spin through air make a good way of rapidly cooling the tube to Room Temperature.

Place slides in array chambers and align cover slip over arrays. Check to make sure the slide is right side up. The spots can usually be visualized by breathing on the slide to fog the surface. Little spots of DNA can been seen as the fog evaporates.

Pipet up 10-12 ul of probe and apply to array:

Regular cover slips:
Pipet probe over the area of the spots and gently place coverslip down, avoiding air bubbles. Fine tip forceps are a great help with this step. (It is worth practicing this step to avoid air bubbles under the cover slip.)

Lifter Slips:
These cover slips have a little edge of teflon along two opposing outer edges (available from Erie Scientific). Place the cover slip over the area of the spots. Place pipet tip along one of the open edges of the slip and slowly pipet the probe out of the tip. Capillary action will draw the probe mix under the slip. Air bubbles are usually not a problem. If they do form, they often go away by themselves.

Chamber assembly and Hybridization

Place the slide in the hyb chamber. To keep the chamber humidified during incubation, pipet 10 uL 3X SSC near one of the edges of the slide, away from the cover slip.

Notice the hydration spot near the left side of the slide
(usually added last just prior to cloding the chamber). 		Lifter slips make pipetting the probe very easy. It wicks
under the coverslip with no bubbles.

Note: the new lifter slips are longer on one side than the ones shown in the picture above, and should be rotated 90 degrees so that the teflon strip is parallel to the long dimension of the slide.

Place the cover over the hyb chamber and tighten the sealing screws finger tight but snug. Be careful not to bump or jar the chamber. You don't want the hydration drop to merge with the cover slip and dilute out the probe, and you don't want the cover slip and probe to bump the side of the chamber and wick out along any surfaces. Gently place the chamber into a 65 C water bath. (Be careful not to burn yourself, if you act swiflty but gently you should have no problem, but putting on two gloves can help insulate your hand from the water). Alternatively you can use some kind of tool or rack for placing the chambers under water, or use another heat source such as a hot bonnet PCR machine, or incubation oven. (The screws are 4-40, so if you use a PCR machine as a heat block, the large knurled thumb screws can be replaced with regular short machine screws.)

If you're doing more than one hyb, it's not a bad idea to mark one of the screws with a sharpy to distinguish the chambers.

Incubate the hybridization 4 hours to overnight. 8 hours seems to be optimal.

Chamber disassembly and washing

Prepare the wash buffers as described below, and have the wash chambers and slide racks, and drying centrifuge ready.

Wash Calculator - Simple JavaScript. Enter the volumes of the washes you would like, and the volumes of the components will be filled in.

Stock Wash 1 Wash 2 Wash 3
20X SSC
10% SDS -- --
H2O
Wash Volume

Carefully remove the chamber and dry it down with paper towels. Water between the sandwich halves can be wicked out with paper towels. If you have several chambers to process, dry them off and remove the screws, but refrain from cracking open the covers until all chambers are ready to open. The time from opening the chamber, to the time the slide rack is immersed in the first wash should be minimized to prevent drying out of the probe. The slide chambers contain a small recess under one edge of the slide for prying the slide out of the chamber. Crack open the slide chamber and place the slide in a slide rack.

The first wash is the hardest

Place the slide rack into the first wash. Gently plunge the rack up and down. The trick is to get the cover slip to fall off without scratching the surface of the slide, or getting stuck on the slide rack. Some slide racks are constructed in such a way that it is very difficult to get the coverslip to fall free of the slide. Wheaton SLide racks are recommended because they contain no obstruction in the vicinity of the cover slip. Be careful not to let the surface of the slide dry at any time during the washes.

Once the cover slip is free, a few plunges up and down are suffucient to wash the slide. Move the rack to the second wash, and third wash with a few plunges each. The accumulated time of all three washes is usually less than five minutes. Even drying of the surface is important to avoid background problems. The centrifuge should be ready with a balance slide rack, and an empty rack padded with a paper towel. After the last wash, place the slide rack into the centrifuge and spin 5 minutes at 500 RPM.

Remove the slides and place them into a dust free box for scanning.

Scanning is currently (Winter/Spring 2000) done in the Serafini Lab. Contact Vivian Peng (penguin@uclink4) or Elva Diaz (eddiaz@uclink4) to see if the scanner is free. Bring a zip disk when you use the scanner. With the current scanning set-up there are two steps to quantifying the arrays. The first is scanning the slide to get a flourescence image. The second is gridding the scanned image to quantify the amount of flourescence at all the spots.