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PCR has stopped working - (Feb/16/2010 )

I have been running a PCR on a nuclear gene from genomic DNA and it has randomly stopped working. I ran a positive control using gDNA that I know is good, with primers elsewhere that I know are good, and I am getting a product, so I know it is not a problem with my thermal cycler, tubes, tips, template or reagents (except primers). I have been using the same primers for months and have been getting a product, and I am using the same stock primers as before, the same taq, the same everything but I can't get a product now. I just get a long smear. Any tips on troubleshooting???


Same template? Same template DNA preparation? Same dilution of template DNA? Have you tried dilutions of your template?


up on what was already said....order new primers? or is something wrong with your thermocycler? And have you checked if the PCR-programm was changed; there are people who just change annealing temps or times if they quickly need to test new primers etc.


Perhaps organic compounds during DNA isolation ended up in your PCR reaction mix. Have you performed a 260/230 test?


As I said before, I am using a DNA template that I have gotten to work before under the same conditions, so I'm not sure whether changing the concentration will help, although I can try. Also, my thermal profile has not changed, that was the first thing I checked. A260/230 is 1.93, although I'm not sure how to interpret this?


I had this problem for a whole week. Easiest way to solve it:

1. New Sterile Water
2. New dNTP Mix
3. New template
4. New Polymerase (if other PCR's don't work)
5. Clean pipettes in 70% ethanol and then wipe down. Or autoclave

When I went with all new materials my PCR's started working like a charm.

-Stuck with Ligation-

Ok I'm running a PCR with all new reagents and I'll see what happens. I don't think its the pipets because I have been using filter tips which have not helped, and no one else in the lab is having my same problem. I'll let you know.


Don't worry, 1.93 is very good. 2 is optimal, <1.4 is poor, although PCR can still work, depending on the manufacturer