cohesive-end ligation - cloning a fusion protein to a vector (Jul/20/2008 )
My assignment is to clone 2 genes into a vector so that it expresses both my gene and the coding sequence of tag.
for gene 1, i am choosing Xho 1 at 5' end and Ecor 1 at 3' end which do not cut in my gene.
Now, I need to syntheize the primers with the sequences of Xho 1 in fwd and EcoR1 in reverse primers, how to determine that how many extra base needs to be added after the restriction enzyme sequence so that when the vector and insert has bee cut by the enzyme and ligate, should not disturb my ATG codon of my gene?
Is there any example/digram which can show me the same or any link which can make me understand this concept.
Once i will understand this , i will try for the second gene.
Many thanks in advance,
This should be useful: