homozygous isolation in Arabidopsis - T-DNA insertion lines (Oct/02/2004 )
hi,
I got this strange problem. maybe somebody has an idea.
I ordered a T-DNA insertion line from Salk institue. they have a collection of T-DNA insertion in Arabidopsis genome. when you recieve the seeds they are not homozygous and it is my job to do it. I have to grow plants from seeds, extract DNA and perform two parallel PCR.
For the first PCR, I use primers that were designed from sequences flanking the suspected insertion point. For the second PCR, one primer was designed from the left border of the T-DNA insert and the second primer was designed to complement genomic sequence that border the expected T-DNA insertion site. Information regarding the T-DNA insertion site and the orientation of the insertion is provided by Salk but is not precise and should be dealt as such.
If I get two PCR product (in the right length) from a plant DNA I conclude it is heterozygous. If I get a product only from the second PCR then I conclude it is homozygous for the insertion. If I get a product only from the first PCR I conclude it is w.t.
I have succeeded in this procedure for two Salk lines. but the third line does not give me homozygous (after 3 generation, 49 plants tested). the thing that does not make sense that I dont get w.t plants either. one possibility is that I have w.t DNA contamination but it is very not likely because I am working on different lines on the same time and on this line I am dealing for 3 generation. I am looking for reasonble possibilities and I'll be happy to get some advice. If you got till here I thank you for reading.
thanks a lot