PCR restriction site - (Jun/17/2001 )
Can anyone give me a quick tutorial on making restrictions sites using PCR? What is 5'-end overhang?
Its putting a restiction site at the 5' end of your PCR primers so you can clone the product into a vector.the key is to add some extra bases after the restiction site so it cuts efficiently. I like to add at leats 6 but others may say as little as 2 extra bases.Check out the back of the NEB catalogue as it has some good info on what enzymes need what extra bases.
I saw your question posted. You just add a restriction site sequence in the 5'end of the primer with some 4-5 extra bases in the 5'region. You can refer some books for those bases or you can add randomly. Because many of the restriction enzymes do not recognise the site which have some 1 or 2 bases around the restriction site. So, it is better to add some 4-5 extra bases in the 5'region in your primer
Best of Luck,