I would like to clone a gene which codes for restriction enzyme Sau 3AI but I am not getting the primer sequence. Can anyone help?
First of all go to the NCBI website to find the "Sau3AI" gene in the genome of the organism from which you want to clone it, once you find the gene sequence just design primers to clone the entire gene into an expression vector.
Remember that you need to make primers with restriction site to clone into the vector, and this restriction enzyme must not cut in your interest gene sequence.
E.g (If BamHI and HindIII dont cut the gene, design primers like this):
Foward with BamHI 5´-TTTTGGATCC ___ ATGXXX(first 20 bases) -´3
Rev with HindIII 5´-TTTTAAGCTT ___ TTA(reverse complement of last 20 bases) -´3
Respected Sreejith , i would like to clone gene that is responsible for production of restriction enzyme Sau 3AI.......
Daniel is correct, you need to clone the methylase as well. However your question is a bit unclear because you say you are not getting the primer sequence. What do you mean, you can not determine what primers to use or you are not getting an amplification product?
First of all go to the NCBI website to find the "Sau3AI" gene in the genome of the organism from which you want to clone it, once you find the gene sequence just design primers to clone the entire gene into an expression vector.
Remember that you need to make primers with restriction site to clone into the vector, and this restriction enzyme must not cut in your interest gene sequence.
E.g (If BamHI and HindIII dont cut the gene, design primers like this):
Foward with BamHI 5´-TTTTGGATCC ___ ATGXXX(first 20 bases) -´3
Rev with HindIII 5´-TTTTAAGCTT ___ TTA(reverse complement of last 20 bases) -´3
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