For debugging I use a stand-alone ST-Link V2. The debugger is connected to an adapter from the ribbon cable to a Molex Pico-Blade connector. By another ribbon cable my board is connected.
I tried another thing: I supplied my board from a 5V external source and unsoldered the AC/DC converter. With this configuration the debugging works every time. But if I unconnect the debugger from my board and power up again (still supplied by the 5V external source) the application doesn't start up. I have a nRF24L01+ module connected via SPI. When the communication between nRF24 and micrcontroller works, a LED should flash two times. In debugging mode it works, but with no debugger connected it doesn't. When I try to connect a logic analyzer (Saleae Logic 8) to the SPI port to check the communication, it works again. I tried to remove the connections between logic analyzer and my board until it doesn't work anymore. The result was, that SCK and GND need to be connected (the logic analyzer doesn't need to work, but must be connected to the computer). When only GND or SCK are connected it doesn't work.
For better understanding of my board I added screenshots of the circuit diagram and layout. In the first design the board was a 4 layer board with seperate GND and 3,3V planes, but now its redesigned to be made an my milling maschine. When the board works again, I will made it a 4 layer board again.
