We have typically recommended a ratio by weight of 20:1 substrate to Trypsin, therefore in this case add 10 ul of 100 ng/ul Trypsin stock solution. For proteomics and large scale applications, we regularly use a 50:1 ratio. Some experimenters use ratios as high as 100:1 so as to limit Trypsin fragments as contaminant in the analysis. Care must be taken when using low amounts of Trypsin that a complete digestion is still obtained. A 10:1 ratio of sample: Tryspin would be the highest amount of trypsin that should be used.