First, for my new sample, I did 100 ng of DNA/25 ul reaction (as it always worked before) and no bands showed up. I then did a test and used 25 ng, 50 ng, and 150 ng each in a total volume of 25 ul. Only the 25 ng tube showed a single band (which was in the correct place also) on my 1.5% agarose gel, while the 50ng and 150ng showed no bands. I thought this was great so I did 4 reactions each with 25 ng / 25 ul reaction. NO BANDS showed up on this run. I did the 25 ng, 50 ng, 150 ng test again, and the same thing happened as before.......one aweseome single band for the 25 ng tube, and no bands in any of the other samples. I then tried a sample that worked previously and that worked fine, so my reagents are most likely just great, but I can't get multiple reactions to work. Any thoughts?
Wow, that sounds incredibly confusing.
So when you do the concentration test you get a band with 25ng of template, but not with 50 or 150ng.
However, when you set it up again using 25ng but with 4 separate tubes, none of them work. Is that correct?
Are you sure the band is the correct one you are looking for? I know you mentioned you are running a 1.5% gel, are you trying to amplify a very small product?
Did you use the same thermal cycler and conditions all three times? Did you use a different polymerase? Did you use the same wells in the thermal cycler? Maybe your block is not heating evenly and some wells are working better than others. If your PCR is not very efficient, this might cause problems.
It sounds like something strange must be going on.