Double Stranded cDNA Low Yields - DS cDNA RT Yield Troubleshooting (Jun/04/2008 )

Hello all-

Currently, I am attempting to establish a method for synthesizing double-stranded cDNA for microarray analysis on a bacterial mixed community. I have attempted the Nimblegen method that utilizes a modified form of the Invitrogen ds cDNA kit and have been frustrated at the low or no ds cDNA results. The work flow has the culture being pelleted, run through Qiagen RNA Easy extraction, DNAse I treatment, Ambient RNA analyzer, Superscript II RT first strand synthesis (with random hexamers), DNA pol I + T4 DNA Pol second strand synthesis, P:C:I extraction, phase lock tube capture, ethanol wash, and then speed vac.

Has anyone in the community attempted this method and encountered similar problems? Also, does anyone know of specific chemicals that could be poisonous to DNA pol I?

I am not sure if this is the correct part of the forum to post this in, I apologize if it is not.

Thank you all for any suggestions.

Thank you.