Probe Check

You can check your probe to see how it looks by scanning it in a spectrophotometer from 200 nM to 700 nM. If you have a cuvette that can read small volumes (i.e. 50 uL) you should see three distinct peaks.

Probe from 2 ug yeast mRNA for each sample. Good RT reaction, with very good Cy conjugation. This probe lit up three arrays very brightly.	Probe from 2 ug yeast mRNA for each sample. Less cDNA and less Cy conjugation, but still very good probe.

It's important to clean up the unconjugated dye, or unincorporated nucleotides effectively. Small amounts of unincorporated dye can have a large absorbance and thus give misleading results.

• As a rule of Thumb, a hybridization will require a minimum of 20 pmol of Cy dye.
• You can clean a cuvette by soaking it for 1 hour in a 1:1 mixture of methanol and concentrated HCl, and then rinse it thoroughly.

You may want to calculate the percent incorporation of Cy dye in your probe.

This probe was made from 15 ug of Total RNA from human cells, and successfully lit up an array. The Cy3 and Cy5 absorbances are small but detectable.

Protocol Notes

• A primary problem with this protocol is assessing the quality of the conjugatable dyes from Amersham. While they are usually good when first opened, they are sensitive to moisture with a short half-life in aqueous environments. Thus take care to store aliquots of them dessicated, under vacuum if possible.
• Troubleshooting the dyes - One could assess the reactivity of the dyes for conjugation using a simple test using a custom oligo, that has amino-allyl groups, or primary amines, spaced at least 8 bases apart (other wise quenching of the dyes can occur: see http://www.phaget4.org/Lib/?field=abstract&what=quench). To see if a given batch of dye is still good for conjugation, one could test it directly by using an aliquot of dye to conjugate to the diagnostic oligo (an ordinary protein like BSA might also work, as the dyes are intended for labeling antibodies). The reaction is very quick, and can be put directly on a gel filtration column (a spin column would do). If two colored bands appear, then you know the dye is conjugating properly (one sees free dye, as well as higher molecular weight species). If one sees only one band, the dye is not conjugating. One could even measure the results spectrophotometrically. In fact one could use such an oligo to measure the kinetics and trouble shoot or optimize every aspect of the conjugation reaction.
• The ratio of aa-dUTP to dTTP mentiod in the protocol is not hard and fast. You may need to optimize the ratio for your particular organism.
• Carbonate Buffer should be made fresh on a regular basis (i.e. once a month).

Chris Seidel, Fall 1999