Dear all
I faced a confusing task. I used pimer LP and RP download by http://signal.salk.edu/tdnaprimers.2.html website for verifying the T-DNA insertion of WiscDsLoxHs. The PCR results was consistent in the previous three PCR tests(95°C for 3min to denature template and activate hot-start enzyme; 28 cycles of 95°C for 15s, 58°C for 30 s, 72°C for 1 min,extend 5 min at 72°C),which showed insertions in both chromosomes. However, when I increased the cycle to 32 and add more(1.25 times) primer in the fourth test, the results showed that one of the pair chromosomes with insertion. I was confused about that, so I use the primer-blast function in NCBI, which showed the LP+RP primers may pair the other locus. May be a non-specific amplification？
Thanks a lot!!!!
What does your no template control look like? A sample amplifying at a high cycle number could be caused by low level of pcr contamination of one of your reagents
Just as a heads up, about 20% of T-DNA lines have a chromosomal translation associated with the insertion site (double-stranded break repair pathway).
If you are seeing unusual segregation during genetic crosses or pollen lethality in heterozygous plants, that's probably why.
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