You may be able to find a plasmid with a cDNA containing your amplicon somewhere on AddGene (https://www.addgene.org). If not, you may be able to clone it the old fashioned way (RT-PCR, then PCR with adapter primers, then ligate into a plasmid of your choice). Beware that once you have it, that plasmid could become a very pernicious contaminant in your RT-qPCR analyses. Due to the very high molar concentration of the target sequence in purified plasmid vs genomic DNA, even a trace amount of plasmid contamination will make a sample seem positive.

Hi,

in addiiton you can order gBlocks with your desired sequence from different vendors like Eurofins. Still would always try to get isolated DNA for the fungal strains as this will better mimic the complex situation which you will have in a real sample

Sven

In general, you don't order these, you have to make them yourself. If you are lucky, someone might have already cloned your gene of interest and used in in a publication and you can contact the author to ask them to send you their plasmid. But you'll most likely need to make it yourself.

Here are the basic steps:

• PCR amplify the region you are using
• clone into a plasmid
• sequence the insert to make sure it's correct
• transform into cells
• grow the cells
• save glycerol stock of colony with your plasmid
• extract the plasmid DNA
• digest to linearize the plasmid
• calculate the copy number concentration
• use serial dilutions to create your standards
• aliquot and freeze serial dilutions

THEN you are ready to test your primers with your dilution series.

Good luck!