qPCR and plasmid positive controls - Why use plasmid positive controls? Are they the best? If so, are there (Aug/29/2007 )
We are doing qPCR and standard PCR to test for a type of fungus in samples. We just got our positive control culture of the fungus in. Another lab used plasmids as their positive control. My questions are.. Why would we want to use plasmids for a positive control rather than pure zoospores from culture? Someone told me that plasmids are more stable and consistant with regular freezing and thawing..but why would there be a difference? Are there any other ways to make a positive control?
If plasmids really are the best choice for a positive control, can anyone tell me how to make plasmids from a fungus culture? Is there a kit that we can use?
Any help would be greatly appreciated!
The reason why people use plasmid controls in qPCR reactions, is because they can accurately measure the amount of DNA, and thus, copy number of the amplified gene within the plasmid which in turn allows for absolute quantification of the target in your experimental samples. If you have your gene of interest cloned within a plasmid, great! This can be used to setup a standard curve, if not you will have to do a relative standard curve based upon serial dilutions of cDNA, gDNA isolated from your tissue. You results will then be measured against your standard curve and give you relative results.