I am attempting to amplify out a 1375bp segment of gDNA and I'm wondering what the ideal PCR parameters would be. On my first attempt, I ran a touchdown pcr and subsequently ran the samples on an agarose gel. I ended up seeing what I interpret as 3 different fragments that seem to sum up to my amplicon size, which I'm thinking could possibly point to my initial annealing temperature being too high? Any insight on my result and tips for my next pcr attempt would be very helpful, thank you.
Seeing multiple bands when you only expected one means your annealing temperature is too LOW. Try a gradient of annealing temps and see if the smaller band disappears.
I assume you mean that you are trying to amplify a 1375 basepair (bp) not 1357 kilo bases (kb).
Unless you are incorrect about the size of the bands in your ladder, NONE of your PCR products are the expected size. The largest is about 700bp
The next size is just under 500. The "band" below the 250 ladder is probably just extra primers.
PCR does NOT give "fragments that add up to the expected product". That's not how it works.
You should go back and double-check the band sizes in the ladder AND the expected PCR product sizes. Something is not matching up as expected. And go talk to a mentor in the lab about the basics of PCR.
Katie A Burnette Thank you for the insight! Forgive me if I'm interpreting this incorrectly, but when you say seeing multiple bands corresponds to annealing temperatures being too low is that because the primers will bind non-specifically? Do you think that's a possible answer to why I'm seeing multiple bands?
Also, I've read that when running a PCR reaction the annealing temp should be around 5°C lower than the Tm of my primers. My primers have ~63°C Tm so should I set my gradient to be from 58°C to 63°C?
I agree entirely with Katie A Burnette .You have 2 problems which are primer related. The smallest band looks like prime dimer so use less primer and a hot start enzyme. It is also essential as she says to run an annealing temperature gradient of 58 to 65. You must also check your primers which are amplifying the wrong size fragments very well so please check your primer integrity because you may need to design new primers to amplify your product
Katie A Burnette Paul Rutland Update: looks like the annealing temp was the problem and not my primers. Ran a TD-PCR with higher annealing temp and got bands in my target range. Thanks for the advice!
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