Enzyme selection for BioBricks digest

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Revision as of 12:34, 8 October 2011 by Michael A. Speer (Talk | contribs)
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Contents

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.

The Standard BioBrick 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---A---S--P-----
+
-----E--X---B---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.

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