Multiple Signals Problem in Sequencing Result

I have a 700bp ITS PCR product. What can I interpret?

I recently performed PCR and observed peaks for HEX and FAM, but the ROX signal was flat. I am unsure how to interpret and present these results.

Could someone please arrange a Zoom meeting to provide detailed guidance on this analysis?

Thank you for your time and assistance.

I have a gene with four different known transcript I`d like to quantify via qPCR (TaqMan). The gene locus was sequenced and the different known transcripts of the gene were found in the sequencing data. So I used the sequence to design specific primers (products size 118-143bp) for the exons that are unique for the isoforms and also one universal primer pair targeting an exon which is present in all variants. I designed all of them to be exon-exon spanning, except one, where it was not possible. The primers were tested for specificity in silco and also the PCR products were analyzed with the BioAnalyzer (DNA 1000 kit). Just the primer pairs that show a specific band of the expected product size were used for qPCR (qPCR conditions: 95°C-20sec; (95°C-1sec, 60°C-20sec)x40)

After carrying out the qPCR triplicates for each sample (4 samples from three different donors), I got this result:

The universal primer is lower expressed than the isoform-specific one. (The CT value for the most expressed isoform is on average 2 cycles lower than for the universal primer)

As they all target different Exons along the transcript I thought about a 3'-end bias in poly-T priming. But the cDNA synthesis was carried out using the Qiagen QuantiTEC kit, which includes a mix of oligo-dT and random primers, which should prevent or minimize the bias.

Furthermore I`m thinking about sequencing the products to make sure that the products match exactly the transcript I want to, even though the BioAnalyzer results were specific.

Does anyone have an idea what might be wrong or how to interpret the data? Or can help me with further ideas of what to inspect.

Thanks in advance!

Recently, a white fungus has appeared on my arabidospsis plate culture and I would like to identify what type of fungus it is (attached photo). Does anyone recognise what type of fungus it is or how I could identify it? Thank you.

The internal transcribed spacer is a molecular identification method used to identify fungi, my question is:

Can I use this method to identify Fusarium oxysporum species complex?

I mutated the U3 region in one subcloning and am trying to add it to wildtype R and U5 regions of HIV-1. To do this, I added an 8 base pair restriction site between U3 and R in order to get it into the same plasmid. I am wondering, would this 8 base pair restriction site have an affect on the overall function of the LTR? I am cloning this all upstream of a reporter gene to see what affects mutations in just the U3 region have on the promoter of the virus. But I am wondering if this 8 bp insertion has any impact of its own? Please and thank you.

EDIT: I do not want any effect from the insertion of the restriction site. I just do not know any other way to combine the mutated U3 region to the wildtype R and U5 regions without the introduction of restriction sites using primers and PCR. I am just wondering if doing so somehow affects the function of the LTR as well? And whether since it is an 8 bp site rather than a 6 bp site for example, that it would have any other impact due to reading frames etc?

I would like to ask whether Ganoderma boninense produces any spores when it is cultured on PDA plates?

To amplify DNA by using PCR Primers are very essential, am currently working on the molecular characterization of some macro fungi , kindly suggest me some suitable forward and reverse primers.