Hi everyone, I have extracted DNA from a biogas bioreactor using Qiagen kit and prep cDNA library then used this library as template to optimize primers for qPCR (taken from papers). Some of the primers hasn't worked and some of them giving different size PCR product. What could be the reason?
Extracted RNA conc.: 50-100 ng/ul
Conc. used for cDNA: 5ul of RNA (50-100ng/ul) into 15ul MM= 20ul reaction vol.
cDNA conc.: around 2000ng
Diluted cDNA conc.: 100ng/ul
cDNA conc. used for primer optimization: 2ul cDNA (100ng/ul) template into 12.5ul PCR MM with 0.5ul each primer set and NFW= 25ul reaction vol.
Couldit be that your dna template is polymorphic for the gene of interest or that the gene has pseudogenes leading to small differences in size of the amplimer.? If the size difference is large then it may just be inappropriate binding of the primers elsewhere than at your target and increasing the annealing temperature may help although in that case you should be getting 2 bands
Never trust primers from a publication, always do your own full set of controls first.
Start with aligning the primers to the organism's genome to see if they bind in more than one location.
Did you also do a DNA extraction from your samples? If yes, start with regular PCR & a good set of positive and negative controls. Once that is working, then you can start the qPCR.
Good luck!
Appreciate the support and time from both Paul and Katie.
As mentioned, polymorphism could show slight difference in band sizes but in my case difference is quite a lot varying among different stages of samples.
Can I ask Katie A S Burnette, What software would you suggest to check the primer alignment?
I'd suggest designing and testing primers for those genes starting with the genome sequence.
Most primers don't have high enough efficiency for qPCR, so design several for each gene.
Include the ones from the paper too (they might be efficient enough).
Start with gradient PCR & see which one(s) work well at the annealing temperature you'll be using for qPCR. And make sure they don't have primer-dimers.
Next, set up a standard curve + negative controls for each pair that looked good using the gradient. Calculate the % efficiency. 95-105% is an acceptable range for publication.
Good luck!
you can get some different mobility issues with gelred or gelgreen compared with EtBR and mobility can be affected by post staining or pre staining so if using alternative dna running dyes it might be worth running the samples in an unstained gel then post staining at the end of the run if all of your samples seem to be the wrong size
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