Hi, all! I've been struggling with this forever . . . I have 2 different genes that I do PCR genotyping for and there are always super bright obviously-positive bands and then bands of varying degrees of brightness. I attached an example image where you can see about half the samples are very bright and obviously positive, 4 are pretty obviously negative, and 2 are kind of ambiguous (sample 2 and 6). Sample 2 is brighter than 6, but neither is remotely as bright as sample 1, for example. So are these pos or neg??
I ran a known pos (which was very bright) and a known neg (which was about the same brightness as mystery sample 6) and a water sample (which was totally blank). But sometimes my known neg is totally blank!
So do I base my pos/neg decision on the brightness of the known neg sample each time I run? Like, if I run a known neg and it is completely blank, then do I count any sample in that run that has any hint of a band as pos? And if I run a known neg and it has a faint band, then do I count any sample brighter than the known neg as a pos, and any sample lighter than the known neg as a neg!?
And it's all totally random as to whether the known neg has a faint band or not. And I've changed out every single reagent a million times and it usually doesn't help. I've also tried diluting the DNA and it usually doesn't help. And it does this for both of my genotyping protocols (a K5rtTA and a GFP).
I'm using an overnight homemade DNA isol buffer shaking at 55C and then 10min at 95C in the morning.
Any help would be appreciated!!
Edited by Hummingbird2, 20 August 2020 - 03:47 AM.