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.
One common reason for PCR sudden failure is that the primers have degraded, This happens if they are dissolved in water . TE is better as it inactivates nucleases. Use a new aliquot of primer or get them remade. Other possibilities are changing the dna purification method has resulted in poor quality dna ( but the positive control sample would show this) or the pcr machine block is failing ,( see if other peoples pcrs are working on the machine that you use
I would suggest you to include a lot of checkpoints to solve your problem.
1- include a positive control where you use the same enzymes and buffers just the template and primers are different. This will be a control for your PCR.
2- check the template quality and size. run it on agarose gel, if you see a smear then the template has degraded. If it is a plasmid then you should see three bands if it is linear then just a single band. If you are unsure about your template then maybe I suggest sequencing to validate your template.
3- check the expiry of your PCR enzyme and buffers.
4- check your primers.
There are many more checkpoints you can add based on at what step you face issues.
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