is this the correct organism Lara?

https://www.ncbi.nlm.nih.gov/nuccore/FJ196084.1

If so you can design longer primers which may be a better option than raising the annealing temperature as the old primers were probably designed to amplify many species so raising the annealing temperature will stop some species from amplifying which can be either a good thing if the one you want remains or a bad thing if you want all species for phylogenic analysis

Hi Lara,

I just want to know how old your primers are. Often older primers get hydrolysed and their length is reduced, so is their specificity. So try to get a new stock, preferably HPLC purified. I have recently used the same primers for some cladocera samples (Daphnia & Moina) and got very good quality sequences from direct sequencing of the amplicons. However, I did PCR with an annealing temp of 52 degrees C for 45 cycles. Therefore I think you first do a grad PCR and see at what highest temperature you get good amplification. Then you can sequence some of the amplicons so generated before looking of other options.

best of luck,

Sibnarayan

Hi Lara,

I think the suggestions of fresh highly purified primer stocks and running a gradient PCR are perfect. There are also some additives such as DMSO, 7-deaza-dGTP, and others. Another thought is a high fidelity polymerase such as Q5. Designing new primers also has potential but could be tedious. If you do go that route, I recommend Primer3 for designing them. It is old school but give you amazing control.

An additional method to try is to run your gels with a high percentage of agarose (1.5-2% depending on amplicon length). Then run the gels for a longer amount of time at a lower voltage. I have seen "hidden" bands in the past. There is one band until you run it in higher percentage agarose for a longer time and then a second band very close to the first will appear. I have had this problem before and had to run a gel for almost 2 hours at 50-70V (may need a long gel for this) and a band appeared that was probably 20 bases different from my intended band. Never could sequence it, but after a few optimization steps including a gradient PCR it was gone. Good luck!

Hi Lara,

I agree with everything that has been recommended to you. In addition, before to start PCR experiments (although you use primers obtained in papers) I always check primers with programs like "SnapGene" or other similar.

If you will have to redesign primers use primer3 or blast in your desired sequence, as they told you before.

I imagine that you are using a specific extraction kit for mitochondrial DNA or specific treatment of samples for that, to avoid contamination with gDNA.

Another tip.... I always include when running each experiment a positive and negative control of my target. Maybe in your lab there are old samples that you can use for this.

Good luck!