I had a very weak amplification of the mouse fecal DNA, any tips?
You may try a few things to improve mouse fecal DNA amplification. First, you should remove the water-insoluble material in the final DNA pellet, which contains PCR inhibitors. Secondly, you may use 45-65 PCR cycles for the amplification, as there is not much mouse DNA in a fecal pellet. If the amplicon bands are too faint, you may simply return the PCR tubes to the thermal cycler for another 45 PCR cycles. Thirdly, you may add 0.5 mM DTT to the PCR reaction, which helps re-activate the inactive polymerase. Fourthly, you may add 0.1 mg/ml BSA to the PCR reaction, which may chelate residual PCR inhibitors. Other usual factors affecting amplification, such as annealing temperature, Mg2+ concentration, primer concentration, etc., also seem to be more critical for successful amplification of fecal DNA then tissue DNA, due to the lower template concentration. Finally, a gel imager may be needed to pick up faint amplicon bands that the naked eye may not see clearly.
