I am working on the T-DNA mutant line. I germinated seeds for about 7 days and genotyped all seedling except for the ungerminated seeds. All seedlings are wild type and heterozygote plants. T-DNA insertion might have caused the seeds to dye. The ungerminated seed has a different phenotype to others. I would like to confirm T-DNA insertion from those seeds. Does anyone have any advice for me?
Thank you so much.
The CTAB method can work for this purpose (attached). Make sure to use linear poly acrylamide so that you get a pellet that you can see. Also grow seeds from your heterzygous plants to see if you still get no homozygous plants. Preferably grow seeds on MS plates because some mutants cannot germinate properly on soil but may germinate if they have some sugar to help get them started.
My suggestion is to isolate genomic DNA to carry out a Southern assay (the most recommended) to determine the T-DNA insertion into the genomic DNA. And, perhaps to perform a PCR assay to amplify your T-DNA and then sequence to confirm it.
Regards
If you are using SALK lines use the following site to design primers to test for the TDNA;
http://signal.salk.edu/tdnaprimers.2.html
If you have heterozygous plants take them to the next generation.
If homozygous seeds are more dormant you should see segregation in germination.
If they are dead look at segregation in the silique - assuming you are using Arabidopsis.
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