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How should I optimize my PCR - (May/19/2012 )

My reaction content:
H2O 8ul
master mix 10ul
forward primer 0.5ul
reverse primer 0.5ul
template dna 1ul

Expected amplicon size: 284bp

First gel image:
Lane 1: 100bp DNA marker
Lane 2: 54.3'C
Lane 3: 54.3'C negative control
Lane 4: 55.5'C
Lane 5: 55.5'C negative control
Lane 6: 56.4'C
Lane 7: 56.4'C negative control
Lane 8: 57.5'C
Lane 9: 57.5'C negative control
Lane 10: 58.3'C
Lane 11: 58.3'C negative control
Lane 12: 60.1'C
Lane 13: 60.1'C negative control

There seemed to be contamination (because there are bands in negative controls)
So I changed the water source.
And since the higher the temperature, the more specific my expected band is, I decide to optimize with higher temp
Here is what I got:

Lane 1: 100bp DNA marker
Lane 2: 61.3'C
Lane 3: 61.3'C negative control
Lane 4: 62.1'C
Lane 5: 62.1'C negative control
Lane 6: 63.1'C
Lane 7: 63.1'C negative control
Lane 8: 63.8'C
Lane 9: 63.8'C negative control
Lane 10: 64.6'C
Lane 11: 64.6'C negative control
Lane 12: 65.2'C
Lane 13: 65.2'C negative control

Even at the highest temp tested so far, there are some nonspecific bands present
So how should I optimize my reaction?
Should I continue to increase the annealing temp? Because if I were to do so, annealing temp will be very close to extension temp. So the 2 reactions may occur together.
Please advice. Thanks.


2-step PCRs are not that uncommon; just remove the annealing step.

Try a Mg2+ gradient (if you are using an enzyme dependent on it).

You don't need a negative control for each temp, just for the lowest one - this is where you will get the most non-specific binding...


Based on your really, really bright bands at the correct size, I would use less starting DNA or fewer cycles.

What are your cycling parameters? With a 284 bp amplicon you can extend for 20-30 seconds even with low quality Taq, less for better Taq. This may help limit formation of the bigger, non-specific bands.