PCR troubleshooting - Primers (Jun/22/2005 )
Okay, I recently prepared two new primers using unopened autoclaved ddH2O. I have prepared a 500 uM stock and a 20 uM working stock which I use in my Master Mix.
I used the pair to amplify an SSR site. This worked very nicely the first time.
Slowly, I have noticed that amplification has consistantly decreased in intensity the last several times I have used them. Now I cannot even get them to amplify.
I re-prepared the 20 uM working stock and I still cannot get any amplification. I know this is not a problem with the water, buffer, dNTPs, enzyme or thermocycler because I can run the same reaction using different primers and it works fine.
Are their any experiments I can do to define the problem? One of my co-workers would like me to check the 'DNA' concentration using Nanodrop. I am not sure this would give me any information even if they were degraded by a nuclease.
Are you sure your template is not the problem. If you think nuclease is your problem, try running you primers on a PAGE. i think 7% tris acetate should work
Hope you get the answer to your problem. All the best
I used the pair to amplify an SSR site. This worked very nicely the first time.
Slowly, I have noticed that amplification has consistantly decreased in intensity the last several times I have used them. Now I cannot even get them to amplify.
I re-prepared the 20 uM working stock and I still cannot get any amplification. I know this is not a problem with the water, buffer, dNTPs, enzyme or thermocycler because I can run the same reaction using different primers and it works fine.
Are their any experiments I can do to define the problem? One of my co-workers would like me to check the 'DNA' concentration using Nanodrop. I am not sure this would give me any information even if they were degraded by a nuclease.
When preparing stock solution, don't use water, instead, use TE buffer and store them in -20C or -70C. Primers tend to degrade in water. If nothing else is wrong, you may have to reorder your primers.
I also read somewhere that, for an unknown reason, some primers tend to degrade very quick while others stable for months or even yrs (if at -20).