help with degenerate primer design - (Jun/23/2008 )
I am working on a protein present in a frog skin.the amino acid and nucleotide sequence of this protein from the frog species is not known. I have identified its presence in the skin by western blot by using a polyclonal antibody.The amino acid sequence of this protein is conserved across various species and i am using this information to design degenerate primers.
The number of primers thus designed are too high:256 for forward and reverse each.
How do i scale down the number of primers?
Please give your suggestions.
thanks and regards,
Hallo , did you chack codon preferences in frog? Some codons are used preferentially so you can eliminate frog nonused from primers design. I suggest you know aa sequence .
I am not sure if this is aplicable for eucaryotes, but in procaryotes it is possible to isolate polyribosomes bind to mRNA with nascent polypeptid. You can use your polyclonal Ab for selection of right polyribosomes and than clone cDNA from mRNA and you get it.
Or another way is to create cDNA library just isolate complete RNA from skin than reverse transcript just mRNA by oligoT primers clone it to expresion vector transformate. grown colonies print on the membrane and use antibodies to identification of the clone harboring your protein