I am genotyping by amplifying a segment from gDNA. I had non-specific bands in my PCR so I did a gradient annealing temp for 30 cycles. Weirdly, the lowest temperature actually seems to give fewer non-specific bands - as you can see in the screenshot where the annealing temps are written above each lane.
This pattern replicates across gDNA preps from different animals.I have triple checked that I loaded lanes in the correct order/oriented tubes correctly in the PCR machine. We know the most intense band here is the correct one by sequencing.
Primer Tm ~ 60C
Mastermix used = Thermo Fischer DreamTaq
Amplicon size ~ 1.8kB
Has anyone seen this or have any idea what could be happening? It goes against most PCR troubleshooting logic that I know.
- It almost looks like a difference in loading
- What is pertinent is that your specific band is MASSIVELY more expressed than those other very minor spurious bands
- Indeed if you were to reduce exposure you would not even see them
- Its about S;N not absolute signal: And your signal to noise is excellent
- Thus, you could take any of those annealing temp and they would be fine
- Your primers are working well
- I suspect you are worrying over nothing of significance
at risk of offending is there any chance that the strip of tubes has been reversed prior to loading and the dirty amplifications are actually the lowest temperature?
Hello
It may happen, as I did the same experience. It might be explained with: Your specific fragment binding to to the primer has a higher efficiency.
Although, I can see from the image that you might not loaded the product equally in the wells. Always for the first gel, load a duplicate next to each other.
Good luck
- Very good Paul
- we all make mistakes and yes that would be the most logical explanation!
- But true or not S;N is such that I would not personally worry; especially with options as to which temp I pick!
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