Happy to help! I used Salk T-DNA lines for my Ph.D and can give you some practical advice.

First, the T-DNA is inserted through a double-stranded break and repair mechanism, which is "messy". The left border can be truncated or expanded during insertion. Also, you will very often have concatenated/truncated/multiple T-DNA pieces all in one "insert". As you might know, the "right T-DNA border" is often truncated or missing and that's why most folks don't use it for PCR.

Your T-DNA of interest could be:

gene_LB----TDNA----RB_gene

gene_RB----TDNA----LB_gene

gene_LB----TDNA----LB_gene

gene_RB----TDNA----RB_gene

etc.

The information about each T-DNA on Salk is an estimate at best. I know from experience that not all lines will even have the insert. If there are several T-DNAs with numbers very close together and locations very close together, that means that they had cross-contamination and only ONE of those Salk lines actually has the insert. Just order all of them.

The location of the insert in/near your gene of interest is also an estimate. Don't be surprised to find that the real insertion is upstream or downstream by 100s to 1000s of bp.

What you should do is:

Design primers that are both "upstream" and "downstream" of the predicted T-DNA insertion site; test combinations of your "upstream" and "downstream" primers with the T-DNA left border and right border primers.

Then Sanger sequence the products to find the exact insertion site.

Classically, folks thought that the T-DNA insertion was too large to get a PCR product across. This is mostly true, but not always.

Also, as a HUGE WARNING:

20% of commercially available T-DNA lines have reciprocal chromosomal translocations associated with the T-DNA insertion event. Some have more than one. You CAN'T get rid of this by backcrossing. You CAN'T detect it by Sanger sequencing or PCR. The only way to know if you do or do not have a translocation is by genetic mapping. See the paper below.

Katie A Burnette

Hi Dr. Burnette, Thanks for taking our your precious time to write me wonderful suggestions. Apart from helping me how to find T-DNA regions you wrote down numerous other suggestions which are really going to be helpful for me in future. I have ordered new primers as you suggested soon I expect to send samples for sequencing to find out T-DNA regions inside my gene of interest.

Thanks again

One other practical piece of advice. Grow a bunch of plants from each batch of seeds. Carefully clip off one cotyledon from each for DNA. Make individual DNA preps from each plant and make a "pooled" DNA tube with a small amount of DNA from each plant. Use the pooled DNA to check for your TDNA in the plant population. Make sure you include wild type DNA and a water negative control. If you find plants with the TDNA, save their DNA as your positive control.

Good luck!