Avinash Sharma

For antibiotic selection, the line should have one copy of your gene insertion if 3:1 ratio in T2. If you want to use PCR, then qPCR.

The traditional method is southern blot. but I think Digital PCR may be a easier way.

I used a 3 primer method for identifying the insertion of my gene in Brachypodium T-DNA mutants , the protocol was given on the T-DNA mutant website.

They must have a protocol for Arabidopsis on the TAIR or SALK website, did you check it?

Briefly it mentions using your Gene specific primer with the forward or reverse T-DNA primer. In one reaction you add both T-DNA forward+ reverse+ your Gene specific primer. I have attached the protocol for your reference, it worked perfectly after the PCR on the gel you will see one band for homozygous insertion and two bands for heterozygous insertion.

Hope it answers the question, feel free to ask any further questions.

Best of luck Avinash Sharma