no aminoallyl-dye conjugation after purification

I need help please. I'm new to microarray and trying to label aminoallyl ssDNA with Alexa555 and 647 but I did not get any conjugation (based on the color of the eluate). I measure the concentration using nanodrop and I can see the DNA is plenty but there is no peaks at the wavelenths of Alexa dyes. Should I see a color on the eluate after purification? Can I use these dyes instead of Cy3 and Cy5 for labelling aminoally? What can I be doing wrong?

QUOTE (lnm06212002 @ Oct 22 2007, 10:15 AM)

I need help please. I'm new to microarray and trying to label aminoallyl ssDNA with Alexa555 and 647 but I did not get any conjugation (based on the color of the eluate). I measure the concentration using nanodrop and I can see the DNA is plenty but there is no peaks at the wavelenths of Alexa dyes. Should I see a color on the eluate after purification? Can I use these dyes instead of Cy3 and Cy5 for labelling aminoally? What can I be doing wrong?

Most amine-reacting dyes require a slightly basic pH (pH8.5-9.0) for coupling, because NH2 in the salt form ( NH3) is not reactive. The other issue is the hydrolysis side reaction of the reactive group in the dye molecule. Hydrolyzed dye is no longer reactive. You need to prepare fresh stock solutions before adding these to the DNA. Always use anhydrous solvent, such as DMF or DMSO. (Sigma sells these). A slight excess of dye to DNA is needed to drive the reaction to completion. Both dyes you have mentioned should reactive with NH2- if they are in the activated form as NHS esters.

QUOTE (genehunter-1 @ Oct 22 2007, 01:37 PM)

QUOTE (lnm06212002 @ Oct 22 2007, 10:15 AM)

I need help please. I'm new to microarray and trying to label aminoallyl ssDNA with Alexa555 and 647 but I did not get any conjugation (based on the color of the eluate). I measure the concentration using nanodrop and I can see the DNA is plenty but there is no peaks at the wavelenths of Alexa dyes. Should I see a color on the eluate after purification? Can I use these dyes instead of Cy3 and Cy5 for labelling aminoally? What can I be doing wrong?

Most amine-reacting dyes require a slightly basic pH (pH8.5-9.0) for coupling, because NH2 in the salt form ( NH3) is not reactive. The other issue is the hydrolysis side reaction of the reactive group in the dye molecule. Hydrolyzed dye is no longer reactive. You need to prepare fresh stock solutions before adding these to the DNA. Always use anhydrous solvent, such as DMF or DMSO. (Sigma sells these). A slight excess of dye to DNA is needed to drive the reaction to completion. Both dyes you have mentioned should reactive with NH2- if they are in the activated form as NHS esters.

Thanks for the reply. The dyes I used are freshly made, I dissolved 1 vial of Alexa in 40 uL of DMSO so I can rule this out. I used 4 uL of the dye per labeling reaction (is this amount enough?). I also eluted the cDNA after purification in 0.1 M NaHCO3 ph9 prior to labeling. My results look like there are no aminoallyl for the dyes to conjugate. I'm using a ratio of 1:4 for DTTP:aa-dUTP, is this ok? Also, I did amplication of the cDNA.

no aminoallyl-dye conjugation after purification - (Oct/22/2007 )