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Chloroplast DNA southern blot - (Mar/04/2007 )

Hi! I'm new here, so first let me introduce myself. I'm a mol. biol. student from Croatia, currently trying to establish southern blot procedure in our lab smile.gif. I'm using Roche DIG DNA Labeling and Detection kit, and + charged nylon membranes. My gene of interest (wich I'm trying to detect in different samples) is rbcL which is encoded in plastid genome. But all southern protocols are for genomic DNA (most for mammalian). So I was wondering if I can use the same protocol here or some changes have to be made? For example, how much chloroplast DNA is there in cell? Do I need to use 5, 10, 20 or more ug of total DNA per lane? Ore less? This is the most important question, because I'm afraid I'm not putting enough DNA on membrane (3-5 ug total DNA per lane). Any advice is more than welcome, because, I've been trying this for a month, and all I'm getting is dirty membrane. I've even tryed dot blot procedure, just to see if something major is wrong, and I am getting a precipitate in the right place so I think probe is OK and hybridization is OK and washing and detection are OK as well, and something before must be wrong...
Thank you very much. M.


Although it is better to using purify chloroplast DNA in sourthern blot, howeve, the total DNA purified from leave tissue which full of chloroplast is still acceptable in such analysis.
Due to high copies of chloroplast genome DNA pruified from green tissue (full of chloroplast), the signal of chloroplast DNA hybridization should be strong and completed digestion is important.