Forgot to mention that I am using Canton S (CS) as my wildtype control. I used another control (w1118) and I tested a separate p-element excision (B101), but I am not really interested in these at the moment. Just the phenomenon of the signal intensities between the Canton S and the B104 p-element excision at the varying temperatures.

Have you tried a gradient PCR to determine the optimal annealing temperature? Primers can have variable efficiencies at different annealing temperatures. Are the primer sites identical between your different Drosophila strains? All it takes is one mismatch near the 3' end of the primer to make your PCR less efficient.

Katie A Burnette Hi Katie,

Yes I did try that, that is why I am having this question. I ran the gradient and at one temperature I am getting a strong wildtype control signal and weak experimental signal and at another temperature I am getting a weak wildtype control signal and a strong experimental signal. The binding sites should be identical, as the excised p-element is nowhere near the binding site. Even if this was the case, wouldn't it be consistent between both the temperatures? As in if the binding site was altered for the experimental B104 line, wouldn't I see a weak signal at both temperatures due to the inability of the primer to bind to the faulty binding site?

When you ran the gradient was it just on one dna or one of each and did you get amplification at both 60 and 62 in the gradient.

The result at 57 looks normal with the smaller band amplifying better but I wonder if at 60 we have 2 effects....1 the longer amplimer which tends to re anneal easily is reannealing less so amplifies more efficiently giving the stronger band and this overcomes the effect of the pcr starting to fail due to the primers not annealing. Of course this idea only has any possibility if the gradient shows that the pcr is likely to fail at 60 and 62

At the temperatures that their cells reside at (as example, human cells the polymerase best work at 37 ° C ), most polymerases work best. At this temperature, however, most of the entire complementary strands will also reattach and interfere with the polymerase function (in cells, the DNA strand is kept unwound during replication by enzymes such as helicase). Some species operate at much higher temperatures.

Paul Rutland Hi Paul,

When I ran the gradient it was with one of each of the genotypes. It's true that for 57C the experimental B104 genotype seemed normal however the Canton S (CS) wildtype control that was used showed no signal. At 60C I again saw no signal with the CS control. Then at 62C there was a very weak signal present for the CS. I included some images below. Thank you for your input, it is greatly appreciated.

Hi Nicholas , with strong amplification at annealing temperatures of 57, 60 and 62 but a weak amplification at 59 I think that the 59c result is a one off result caused by some mistake in the pcr like a leaky tube cap or one reagent not mixing in well causing a poor amplification just in the one sample

Nicholas Narwold

At least your deletion line PCR worked pretty good now, compared to the first image in your 'question'. You have only one deletion line?

We use Promega GoTaqTM DNA polymerase a lot for routine PCR amplification. My impression is that this polymerase is robust for amplifying shorter bands. You are amplifying a band more than 2 kb (and from genomic DNA template, not even from plasmids). That is why your band is faint and sometimes it appears and sometimes doesn't. Promega did not mention what is the longest DNA size GoTaqTM DNA polymerase can amplify, but a 2 kb PCR product probably near its a bility to amplify, and would only give you a faint band on a gel. Promega also sell GoTaqTM *long* DNA polymerase [1], which they advice to use for amplifying > 3kb genomic DNA. I bet the bands would show up nicely and bright if you use a 'long-distance' or more robust DNA polymerase to amplify your control (wild-type, no deletion) samples. Ask for a small amount of robust DNA polymerase from other labs and try it out. Please do update us if you have result (both positive and negative), I would very much like to see if this is the case.

[1] https://www.promegaconnections.com/how-do-i-choose-the-right-gotaq-product-to-suit-my-needs-for-endpoint-pcr/

Its due to specificity of primers.

Yuan-Yeu Yau That may also be a factor. I searched around and found some sources that mentioned that the GoTaq master mix can be used to amplify genomic DNA up to 4kb, but passed 3kb there will begin to be a reduction in efficiency. I will try your suggestion and give you an update when I have more info. Paul Rutland I suppose that could also be a contributing factor, especially when working with so many samples (at the time it was more than 60). However, I do make it a point in my protocol to mix each reaction vessel thoroughly by pipetting up and down and before I add the samples to the thermal cycler I spin them down briefly (5 seconds with less than 5,000 rpm) just so I can be certain that all the solution is aggregated at the bottom of the tube and there is no solution adhering to the walls.

Thank you both so much, and everyone else, for taking time out of your day to help me out. Your input is greatly appreciated.

happy to have been part of the discussion Nicholas