Inconsistent PCR amplification

[Please_Work_This_Time]

Hello, hope somebody can offer some advice.

I am trying to demonstrate the validity of exisiting published PCR amplification methods for the detection of different Mycoplasma species. In order to validate the methods I am assessing the specificity by conducting a true positive detection rate assay using a panel of positive control type strains or field isolates and hoping for a 100% true positive detection rate. If successful, I will perform another specificity PCR assay to assess the false positive detection rate using a panel of negative controls.

I have found inconsistent amplification across the positive control panel and wonder why this may be the case. I used the same PCR master mix, thermocycler, thoroughly mixed all reagents and the master mix after adding each component so I wonder whether it maybe the amount or quality of template which is the reason. Unfortunately our lab doesn't have a UV spectrophotemeter so I cannot be certain that there is enough template. However, the broth that I use to grow the Mycoplasmas has a distinct red to orange colour change during growth, and by this stage there are normally enough bacteria for good PCR product amplification. Another issue might be the length of time the cultures are kept in storage at -40C, I have had problems with type strain aliquots frozen down between 2003 and 2007.

Any advice would be most welcome...
Thank you.

[sunglassesatnight]

First thing I would do would be find someone with a nanodrop and see if you have any DNA in your samples. You only need 1 molecule of template to get amplification (a guy in my lab runs 0.2 molecules per well and it works in 1/5 of the wells) so if there is any DNA then you should get a product. If there is DNA, here is what to do next. Replace or remake all the components of your PCR reaction one at a time. You might have spotty dNTPs, water, primers, or buffer. Only one way to know for sure. Then eventually you should get clean, consistent PCRs. It just occurred to me that you could also try increasing your magnesium concentration or adding some tween20 or BSA to the PCR to stabilize polymerase.