I work with transgenic plants that we transform using agrobacterium. When calli produce tissues, I screen the tissues using PCR. Lately, I have been using thermofisher's Direct PCR. Through the past year, we have had no issues with the kit or with our positive controls.

I have been trying to screen plant shoots since April. These plants were transformed using agro with a PHA vector. There are five separate PHA vectors. For a positive control, we use the appropriate PHA plasmid (i.e. if the calli transformed with PHA 231 produces shoots and we are screening them, I would use the PHA 231 plasmid as the positive control). The plasmid stocks worked well the last time I used them, so confirm PHA vector insertion through a triparental mating protocol. My positive controls were bright and consistent in my agarose gels! This was in October 2021 and November 2021.

Since I have started screening plant shoots, the plasmids are not consistently producing bands. These are the same plasmid extractions used in October 2021 and November 2021. We have extracted new plasmids from E.coli plates and they are not working either. I have prepared reactions for separate PCR machines to determine if there is a problem with the machine. I have run other reactions (with different samples, primers, and positive controls) to see if there is a problem with the machine or my technique. These reactions work fine without contamination. The positive controls show up fine.

Sometimes, the PHA plasmids will produce positive bands. Sometimes, the same plasmid will not produce a band.. I don't know what the problem is.. Any suggestions would be greatly appreciated. I can provide more information or gel photos if needed.