I used to use PCR primers even after 3-4 months and they worked, but now something seems to be wrong with them, they stop working after few runs. I am totally clueless about it.
if your primers are dissolved in water then it may absorb CO2 from the air and become acidic and degrade the single stranded dna of the primer, Best to dissolve primers in TE which is alkaline and has EDTA to stop nuclease activity which also may be degrading your primers
Working primers can be stored upto 4-6 months at -20C and stocks can be stored upto 10-12 months at -80C. If you stored your working primers at -20C, they will work just fine. Check the quality of your DNA and other PCR chemicals are used in proper concentrations.
if your primers are dissolved in water then it may absorb CO2 from the air and become acidic and degrade the single stranded dna of the primer, Best to dissolve primers in TE which is alkaline and has EDTA to stop nuclease activity which also may be degrading your primers
Primers, If stored in -20 or -80 will not degrade for years. I myself have used 8-9 years old primers which were stored in -20 and the results were absolutely fine, even I have checked those primers on 2-2.5% agarose gel, they all look fine and not degraded. Please find the picture attached.
I think you should try making fresh working primers and try PCR. Or you can check the integrity of primers by running on 2-2.5% gel.
Good Luck.
Don't try to trouble shoot this. Primers are really, really cheap. Just toss your regents and try again.
- Badges
- Science method