For a gel that is 14 cm in the running dimension, and 3 cm between each row of wells, apply 200 volts for 15 minutes.

13. Take digital photo of gel and store image for future reference.

The gels should show bands of fairly uniform brightness distributed in size between 600 to 2000 base-pairs as in Figure 2. Further computer analysis of such images can be carried out with image analysis packages to provide a list of the number and size of bands. Ideally this information can be made available during analysis of the data from hybridizations involving these PCR products.

2.5 Purify PCR products

1. Fill 96 well V-bottom plates with 200ul per well of ethanol/acetate mix.

The ethanol acetate solution used for precipitation is less acidic (pH 6) than is typically used. In this instance, more acidic solutions produce precipitates which are harder to resuspend without improving yield.

2. Transfer 100ul per well of PCR product into V-bottom plates and mix by pipetting a volume of 75 µl per well four times.

3. Place the plates in -80°C freezer for one hour or store overnight at -20°C.

Place plates at -20°C if they are to be left for more than one hour, aggressive precipitation produces precipitates which are hard to resuspend.

4. Thaw the plates to reduce brittleness and melt any ice, which may have formed in the wells.

5. Load the plates into a centrifuge with a horizontal microtiter plate rotor and spin at 2600 x g for 40 minutes at 4°C.

6. Aspirate the supernatant from each well using the Immunowash plate washer.

Settings for the depth of aspiration by the plate washer will need to be adjusted to suit the microtiter plates used. It is advisable to leave approximately 10-20ml in the bottom of the well to avoid disturbing the pellet.

7. Deliver 200 µl of 70% ethanol to each well in the plate using the Immunowash plate washer.

8. Centrifuge plates at 2600 x g for 40 minutes.

9. Aspirate the supernatant from each well using the Immunowash plate washer.

10. Allow the plates to dry overnight in a closed drawer.

Do not dry in a speed-vac. Desiccated PCR products are hard to resuspend.

2.6 Resuspend the PCR products

1. Add 40 µl of 3X SSC per well. Seal plates with a foil sealer, taking care to achieve a tight seal over each well.

2. Place the plates in heat sealable bags with paper towels moistened with 3X SSC and seal the bag with a heat sealer.

The high external humidity within the sealed bag helps keep the volumes in the individual wells from varying.

3. Place the bags in a 65°C incubator for 2 hours, then turn off the heat in the incubator.

Allowing the plates to cool down gradually in the incubator avoids condensation on the sealers.

2.7 Check PCR resuspension for yield by fluorometric determination of DNA concentration

Analyze 1 µl of resuspended PCR product from one row of wells from each plate on a 2% agarose gel as previously described. Adequate precipitation and resuspension will produce very intense bands, with no material failing to leave the loading well, and no smear of material from the band towards the loading well.

While it would be ideal to be able to exactingly quantify each EST PCR product and spot each DNA species at equivalent concentrations, it is impractical for most labs to do so when thousands of ESTs must be prepared. Fortunately, it is possible to use a strategy where excess DNA is spotted, so that the exact quantities used do not produce much variation in the observed results. When using this strategy, it is necessary to track the average productivity of the PCR reactions. Fluorometry provides a simple way to obtain an approximate concentration of the double-stranded PCR product in the PCR reaction mix.

Store the plates at -20°C after resuspension.

Materials, Reagents and Solutions