cDNA cloning by PCR - cloning (Mar/17/2005 )
i have purified a protein whose N-terminal sequence is known. If sequence of only Nterminal is known how shuould i design the primers to get the amplifcation of the desired gene can i get a cDNA clone from a single primer .what should be the primer for other flanking region of the gene?
Can I know from which organism that you isolated your protein?
How many base pair that you know?
I need to know those information before I can know what to do.
ive isolated my proteins from outer membrane of Salmonella typhimurium.for one we have sequenced 15 aminoacids and for the other 7.
To get the primer for other flanking region of the gene, I think is good to use bioinformatic software and make use of swissprot protein database.
Now you know 15aa (=45bp). load your know 45bp on NCBI and BLAST. From the BLAST search, the programe will show you a lot of similar protein. You can make use the information from the data base predict your reverse primer.