Basically for my experiment,i will go and collect my raw plant sample from sea for 3 times.I explain what i will do for my first time collection because the other two times it will be same way of analysis (i consider 3 times as a replicates as well).
So for my time collection:
I will process my sample and start from crude enzyme for the analysis:
1) i will do spectrophotometric (colorimetric) analysis in 96 well plate (its just a plate with many wells) .analysis usually with blank, control, 4 replicates of sample (altogether 6 wells for every row)
a) i will have 3 different composition (A,B,C) and do this colorimetric analysis by time course for 20 minutes. Once i get the absorbance i will calculate and plot the result for this 3 different composition (A,B,C) separately, where x-axis will be time (20 minutes) and y-axis as the activtiy. The 20 minutes reaction i will get reading for every 1 minutes interval. So i will include all the starts from 0 minutes to 20 minutes reading in this comparison.Then i want to compare which 3 (A,B,C) will give a good result.
So, from my understanding if 3 samlpe and by tiime course comparison we will be using two way repeated measure ANOVA. But when i tried to analyse using sigmaplot it falied normality test but passed for variance test and analyzed using "Hold-Sidak method" which give me result satisfying for me but when comparnig these 3 (A,B,C) by time course i didn't see any changes from the graph but this statistical analysis shows significantly different(probably minute changes). but this is not acceptable for the reviewer or my supervisor.
Thus, to get a right analysis (normality test passed) what are the steps i should correct? of number of sample? besides that in sigmaplot i dont know how to create descriptive analysis, as just able to get the variance result and pairwise comparison result
b) Next analysis will be same as before, x-axis is time course for 20 minutes and y-axis will be activity. The analysis is with fixed concentration of 3 different substrates (mixed in one assay).This will be one line graph (presumably linear line or linear and then plateu formed).I'm not sure do i need to repeat the analysis 3 times? because when doing the analysis i have 4 wells as replicates? Do i need to do any statistical analysis here, as im not sure.