The last couple of months I've been trying to replicate a small part of the DNA by using PCR. While everything was working great at first the PCR just stopped working one day. I haven't changed anything regarding the components nor the procedure and I really can't figure out what happened and why it's suddenly not working at all. Any help would be greatly appreciated.
That sort of sudden failure is typically due to degraded primers.
Order a new batch of primers, make fresh stocks & try again.
Good luck!
Check the pcr mastermix and integrity of your plasmids. Multiple freeze-thaw cycles could hinder the reaction. Also, it is recommend to check your DNA sample.
One more idea. Double check that no one has changed the parameters on your thermocycler. It has happened to me, someone changed the annealing temperature & saved the program with the same name.
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