multiplex PCR for three sets of primers - (Dec/07/2005 )

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HI, I am presently developing a multiplex PCR system for detection of mycobacteria. Three gene are interested. Each set of primers works well in the individual amplification (please note that I need to run nested PCR for each gene of interested, therefore I have 6 pairs of primers). I had tried equimolar primer method in multiplex reaction for first trial (single run PCR, using average anealing temp.), but it failed.
In 2nd trial, I increased primer conc. to 1.25 micromolar, buffer conc. to 2X, lower polymerase to 1 Unit/50 microlitre; besides changing reagent concentration, I also used nested PCR cycling condition as I did for the individual amplification (two rounds of PCR, average annealing temp)

IS that possible to adpot nested method for multiplex? The reason I used nested is because the 1st round's primer is usually not sensitive enough, especially my samples are normally paraffin-embbeded tissue.

please kinly suggest if any..


Thank you!!

what are your expected size products?