Hello everyone, I have a bit of an interesting and frustrating situation I am currently facing. So to preface this, I have been running a PCR looking at different levels of splice variants occurring in the KCNMA1 gene, exon 23b. The primer sets and thermocycler conditions for this were pulled directly from a paper. I have run this PCR across various samples for the better part of a year and it has worked perfectly almost every time. Recently, I tried to run this PCR again except it didn't work to my surprise. no problem, probably just primer getting old and degrading. So I reordered the primers. This still didn't work so I reordered the mastermix (It calls for using NEB 2x taq, a very basic PCR formula). This didn't help either. Seeing no other options, I remade the cDNA associated with these samples. This STILL did not work. The main issue is I do get low levels of amplification of one of the splice variants of interest, but I do not get much amplification of the other. I will attach pictures of what a good run should look like and a picture of what the runs I have been getting look like. I've been hesitant to redesign primers or change too much about the thermocycler conditions since the primer sets and conditions have worked so well for many runs in the past. It's important to note too that I do include control samples in every run that have worked very well in the past. I've repeated this PCR 15+ times now with very limited success. The strangest part is sometimes I will get a few samples in a run to amplify robustly but I cannot seem to get a full all-around good run that amplifies all the samples well. Today my last-ditch effort was to try a hifi taq polymerase to see if this would help the amplification but unfortunately it did not. I thought the thermocycler may be having issues too, so I tried a different one and got the same poor results. does anyone have any suggestions? I feel like I've tried basically everything I could without dramatically changing the protocol or master mix.
Thank you in advance.
One clear difference is that the primer dimer is very strong on the bad amplification so I am thinking that you have PD contamination of your reagents or pipettes or working area which is amplifying so well that it is removing all the primer so leading to no amplimer of the larger primer set. Do you run a no template control pcr along with your samples, If you do remake primers then make the amplimer larger than these ones to avoid any contamination amplifying
Hi Michael,
I have had a look at your gels. The first thing I noticed is that you have a lot of unused primers or primer dimers (hard to see on the pictures) which points in the direction of non-optimal primer design or PCR conditions.
Regarding your good -looking amplification - I am not sure if you only see 2 bands - there might be three bands (I am not sure about the upper band - have you ever done a polyacrylamide gel to see if you have really only two bands in total?).
I think I have found the paper from where you got the sequences and the cycling conditions. There are doing 32 cycles here but as you do not get too much PCR product and you have still a lot of unused primer I would consider to do at least 35 cycles.
However, this might not help regarding the issue that you do not get the upper band (or two bands) in your recent PCRs.
I think it is good that you ordered new primers - so that is not likely the issue you are facing. You said you did synthesize new cDNA, however have you also prepared new RNA/mRNA for the synthesis?
If not I would strongly recommend to prepare new RNA/mRNA to make sure that the integrity of your template RNA is fine. If you have done this already are you sure you used the exact same tissue as these splice variant seem to really vary between tissue and if I got it correctly even in the CNS itself?
In addition, as I assume you are working with mice, be aware that splicing can also vary between mouse strains (however, I do not know if this is true for the kcnma1 gene). So make sure that you always isolate the RNA from mice with the same genetic background (if you do not do so already).
I would also consider sending you PCR product for sequencing to verify that you are really amplifying the correct product.
To further improve your PCR results I would always consider using a hot start polymerase (or doing a manual hot start).
Thank you both so much for the detailed answers! I wasnt sure how to directly reply to your posts but I hope you see this. I will continue to troubleshoot with priner dimer contamination in mind!
A PCR that previously worked well but has suddenly stopped may be experiencing issues with reagents, temperature control, or primer design. Reagent degradation, especially Taq polymerase, dNTPs, and primers, is a common cause. Additionally, temperature fluctuations in the thermocycler or incorrect annealing temperatures can hinder the reaction. Primer issues, such as degradation or incorrect annealing temperature (Tm), should also be investigated.
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