Enzyme selection for BioBricks digest
Introduction
BioBricking is a method of assembling two pieces of DNA together. These pieces of DNA are called "parts" and are flanked by four standard restriction sites. These sites are chosen such that when two parts are assembled together they retain the original format.
-----E--X---Part---S--P-----
In this case the XbaI (TCTAGA) and SpeI (ACTAGT)restriction sites have compatible sticky ends (CTAG), but when they are ligated together they form a benign "scar" (TACTAG) which is composed of the left section of the SpeI site and the right section of the XbaI site. The key to performing assembly correctly is choosing the correct enzymes.
As an Example we'll make a theoretical assebly of parts A and B:
-----E--X---Part---S--P-----
+
-----E--X---Part---S--P-----
Digests
To place the insert part down-stream of the vector part
Downstream Insert - X,P
Upstream Insert - E,S
Vector with downstream part- E,X
E= EcoRI
X= XbaI
S= SpeI
P= PstI
Notes
Very small (<100kb) inserts have shown to be difficult to remove from a gel purification. It is recommended that during assembly very small pieces be kept on the vector and larger pieces be used as inserts if possible. Otherwise just use 3A assembly or Amplified Insert Assembly.
