I am genotyping a T-DNA mutant in Arabidopsis and I get a band in the T-DNA reaction for wild type (Col-0). I've run this reaction four times now and used different wild type samples but still seen the T-DNA band. Has anybody experienced this before?
PCR reaction conditions:
Initial denaturing at 95C for 3min
Denaturing at 95C for 30sec (35 cycles)
Annealing at 52C for 30sec (35 cycles)
Extension at 72C for 1min (35 cycles)
Final extension at 72C for 10min
W=Wild type, T=T-DNA
My guess is contamination. Include a negative control (water, no DNA). I bet you'll see the same band. You could also try using a different T-DNA primer (e.g. JL202 instead of p745).
I think its contamination, please put up a fresh PCR with appropriate controls (no template control). If you have some previously isolated WT DNA samples, include that as well.
Most probably its the T-DNA primer that's contaminated, change that primer in this fresh PCR. This PCR will give you clarity (no template control + old WT DNA + Fresh T-DNA Primer).
If you still get the same results, contamination could be either because of DNA contamination in your PCR reagents or DNA contamination during genomic DNA isolation.
All the best.
Agree with other comments: A: try to eliminate contamination by using water/extraction buffer control); if that is positive, exchange reactives. B: sometimes the TDNA primers give background. Do you have Salk lines? i think the SALK LBb1 gives more problems (better use LBa1 and increase annealing temperature (58/60 e.g.)).
I had issues with a left-border primer making a product in WT as well. We called it Lb1 (guessing it was the SALK LBb1) mentioned above. The higher annealing temperature strategy helped.
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