I am trying to genotype a SAIL line with the primers designed on the SIGNAL/SALK website. When genotyping known homozygous mutants, I am getting no bands with the LP+RP but a band larger than 1 kb with the LB+RP. I expected the band to be less than 800 kb with the LB+RP reaction. Has anyone seen this before. With the wild-type control, I get a band with the LP+RP and no bands with the LB+RP. I am confident that this is the correct mutant because the plants have the expected the phenotype and I personally received seeds from our collaborator.
Thanks!
Ryan
You can have filler DNA at the LB and/or RB integration site leading to larger than expected PCR product size. To be sure, clone the PCR product and sequence.
This is totally normal for Arabidopsis TDNA lines. The TDNA insertion can be truncated, contain "filler" DNA or even have multiple TDNAs in the same site. It's also normal to only be able to use either the RB or LB due to truncation and/or multiple insertions. If you are really curious, sequence the product. Otherwise, it really doesn't matter and is really, really common. Just as a heads up, so are chromosomal translocations. See attached article for more info about characterizing TDNA lines
Article Chromosomal translocations are a common phenomenon in Arabid...
Agree with Katie - super common for T-DNA insertions not to "be" exactly where you think they are. This is one reason iSect suggests primers some distance (~400bp) from the insertion point. I've also had T-DNA insertion mutants with back-to-back insertions, so that both RP+LB and LP+LB primer combinations give a product! Really normal and nothing to worry about :)
Thanks for the advice and tips! It puts my mind at ease. Happy Holidays!
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