Positive control doesn’t work, sample does, but negative control still negative - I need help fast (Mar/14/2007 )
Okay I’m new to this whole thing but I have a question that I really can’t figure out. I was doing a PCR on various samples that contained different amounts of template. Everything else from the primer concentration to the PCR mix, to even the primer used was the same for each sample. I used a positive control and a negative control. All my negative controls came out negative. All but one of my positive controls came out positive. Now were I get confused at is for the one positive control that didn’t work, I still got amplification results in all my replicates for that sample. What could be going on to cause this? At first I thought it might be contamination but my negative was negative. Then I was thinking maybe it was the primer itself but the same primer was used in the other samples and worked. So does anyone know what it could be?
When you made up your reactions, did you prepare the positive control as a master mix, or did you make up a number of positive controls? Could you have forgotten to add template to one positive control tube?
I made it up as a master mix.
I made it up as a master mix.
OK. If you made up a master mix, and all the others worked, we can positively say it's not a template, primer, enzyme or reagent problem. To my mind, that means a problem in the specific reaction tube. Are you using strips or individual tubes?
Perhaps it's just a case of pure blind dumb bad luck. I'd try it again and see what happens. If all else fails, see the posting about the "gods" of molecular biology. Your offerings are clearly not being accepted.
I used strip tubes...Well I hope this isn't the start of bad luck...I do kinda want to make a career out of this...well I think as of now... 
...Hum maybe a new career move is in the future!
I think this is just an accident. I normally don't give a sh*t on my positive control, as long as the negative doesn't show any band.
There could be millions of reasons why your positive control didn't work, so if this isn't the prime issue, simply carry on.
However, if you do want to make your positive control work, try it again, and do replicates. Sometimes if there is a false hole in your PCR machine and you were so unlucky to pick it, you are screwed up.
Good luck/
Thank you for answering my question. I have no idea (still) why it did not work that time but I did re-run it with replicates using individual tubes and all my positives came out. Maybe if I get any free time I will try an experiment using strip tubes vs. individual tubes to see if that was the root of my issues.