Dear Friends
i am cloning some genes responsible for killer toxin proteins. first i also lated my protein and then did SDS PAGE and checked it mw that was about 68kd.then i get its dequence by mass spectrophotometer than did blast but it doesn't matc with any protein. it has smiliarty with lipase little but some with unpunlished proteind whose function is unknow. my sequnce protein has killing activity agains yeast strains. i read many papers and concluded that the gene of killer toxin protein is on linear plasmid in some yeast but in some gDNA. now i am confues that how can i come to know that my gene is where and how should i start my pcr to clone a gene coding that killer toxin protein. please help me if any body has worked with plasmid etc
Did u try tBLASTn?? Try checking the papers normally the toxic proteins are produced by the plasmid DNA. Try to see wat other toxic proteins are produced by other yeast strains and check where r they located then try to do a multiple alignment of the gDNA or plasmid DNA as given in papers to find out possible location of toxic proteins on the DNA strands then u can check your strain with the similar location.
If nothing works try to reverse transcribe then from the cDNA got try to label it and use it as a probe and find out the location of the gene in the yeast strain.
thanks abhinaya anandamurugan i did bast before now i am trying to clone the gene then i will try and follow your suggestions.
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