I am doing MSP after BS treatment.. I standarized the protocol with different conc. of BS but when I did with my samples, I have faced many problems? I have 6 samples but first time I got in only 2 lanes and again repeated then got in 4 lanes , again repeated it came in all lanes but v. v. faint. for U allele nothing is coming but it is expected to come.. I do not know why this is happening? plz suggest me something? I am attaching the pics...after adding NaOH for denaturation I do pipetting up and down to break DNA.. So another doubt is when after adding NaOH , we do pipetting up and down 200 times for breaking DNA then time for all the samples in NaoH not remain equal. first samples remain for longer time and last one for less time. how can we equalise this time? and what if we do not do this up down?? In 1 paper DNA was heat denaured prior to BS treatment and then 1 ug of this DNA was taken for BS treatment.. Can that we a good method?? and anoher doubt is I am using salmon sperm DNA as carrier DNA and so my doubt is how can we estimate that after BS treatment how much DNA is our DNA of interest and how much is salmon sperm DNA?? plz do suggest some solution???