Hey OP, I don't know if you have fixed your problem, but I have been having issues lately that are very similar to what you experienced, and I discovered some stuff that could also be of importance to you.
I needed to PCR a 3.7kb fragment from a plasmid (10kb) with primers that had 10-15bp overhang. Lots of AT, so annealing temp was low (45-55, according to different online calculators, i picked 50).
I managed 1 successful PCR and got my fragment, but when I repeated it I got only smears, looking almost exactly like the smears you got
Refer to the attached photo, I did the PCR a couple of times and this was the last time it failed. Tested all the Pfx polymerases we had with different Mg2+ concentration.
2, 5, 8 are my blancs, 3, 6, 9 is with 1mM Mg2+, 4, 7, 10 is with 2mM Mg2+. 1,2,3 is with Pfx50, 4,5,6 with Pfx plat and 7,8,9 is with a pfx plat from a different box. All pols used with the buffers , enhancers and mg2+ that came with them. dNTP's and primer mix was the same for all sample
All our mixes without the template are prepared in a prePCR lab.
The I proceeded unscientifically; I changed a lot of things at once.
The template I was using was diluted with MQ that I also used for adding to the blanc (as much as the template, 1ul). I made a fresh maxi of my template plasmid, solving it in MQ that I took from the PCR lab.
I re-made my dNTP's in the PCR lab.
I made new primer solution.
I used to do all my PCR's overnight, then holding them at 16C till the morning when I analyzed on gel. This time I did the PCR and afterwards I immediately put the reactions on gel. I also use Taq platinum polymerase, thinking if Taq can't do it no-one can! I used the manufacturer's protocol and annealed at 50C and elongation time was 3'30". reaction volume was 20ul. I used different amounts of template, 1ul (@800ng/ul), 1/10 dil of this, 1/100 dilution, 1/1000 dilution and 1/10^4 dilution
Now I see bands (at the right height)(see photo 0704: lane2 = blank, 3, 4, 5, 6 10E-4, 10E-3, 10E-2, 10E-1 dilution of template and lane 7 = 1ul = 800ng of template).
Then I make repeat PCR O/n with the same conditions, and also included 2 samples with the old primerstocks, 2 samples with pfx50 polymerase (pfx50 mix made as recommenced, pcr program as used for taq. New primerstock) and in a different block i used same mix (also taq) and different amounts of template but used 34 cycle instead of 30. Photo 0804 is result
Block 1 30 cycles (30"@ 95, 30x(20"95, 30"@ 50, 3'30"@ 68), 2'@ 68, infinite @ 16)
Lane 1 ladder
taq mix new primer
Lane 2 no template
Lane 3 1/1000 dil template
Lane 4 1/100 template
Lane 5 1/10 template
taq mix old primer
Lane 6 no template
Lane 7 1/100 dil template
Lane 8 No template
Lane 9 1/100 dil template
Block 2 34 cycles (30"@ 95, 34x(20"95, 30"@ 50, 3'30"@ 68), 2'@ 68, infinite @ 4)
Lane 10 ladder
Lane 11 1/10000dilution
Lane 12 1/1000 dilution
Lane 13 1/100 dilution
So yeah basically for me it doesn't really matter what template dilution I use, or how many cycles I run. It also doesn't matter if I leave it overnight (it seems to even improve signal strength of lower dilution bands). Pfx50 does not do what it's supposed to do: there's smears in both lanes and no band. I also don't see any primers? I am curious about the contamination; I will do the taq PCR again this time adding different components of the Pfx50 mix to it (basically only two; the Pfx50 and the Pfx50 mix), with and without template. Also, next week I will test Phusion polymerase I got from a friend, and we got new Pfx platinum that I will also test.