I'm trying to do something like this
Lines = file.readlines()
# do something
Lines = file.readlines()
but the second time Lines is empty. Is that normal?
Yes, because .readlines() advances the file pointer to the end of the file.
Why not just store a copy of the lines in a variable?
file_lines = file.readlines()
Lines = list(file_lines)
# do something that modifies Lines
Lines = list(file_lines)
It'd be far more efficient than hitting the disk twice. (Note that the list() call is necessary to create a copy of the list so that modifications to Lines won't affect file_lines.)