Enzyme selection for BioBricks digest

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'''A BioBricks part:'''
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==Introduction==
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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. 
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*-----E--X---Part---S--P-----
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<center>'''The Standard BioBrick Format'''<br>-----E--X---Part---S--P-----</center>
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Vector with upstream part - S,P
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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. 
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Downstream Insert - X,P
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As an Example we'll make a theoretical assebly of parts A and B:
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<center><br>-----E--X---'''A'''---S--P-----<br>'''+'''<br>-----E--X---'''B'''---S--P-----</center>
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Upstream Insert - E,S
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==Amplified Insert Assembly & Standard Assembly Digests==
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Both amplified insert assembly and standard assembly operate on the distinction between the vector part (the part which will stay in its plasmid) and the insert part (the part which will lave its plasmid and be placed in the vector plasmid next to the vector part).  With these methods either part A or part B could be the vector or insert.  If you're performing Standard Assembly there is an advantage to making the larger part the insert because it will be easier to gel-extract.  If you're performing Amplified Insert Assembly there is an advantage to making the smaller part the insert because the PCR amplification step will go faster.  For our example we'll choose part A as the insert part.   
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Vector with downstream part- E,X
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===To place part A upstream of part B===
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*Part A - EcoRI & SpeI (insert)
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*Part B - EcoRI & XbaI (vector)
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===To place part A downstream of part B===
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*Part A - XbaI & PstI  (insert)
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*Part B - SpeI & PstI  (vector)
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==Three-Antibiotic Assembly Digests==
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In 3A assembly both part A and part B will be inserted into a third plasmid with a different antibiotic resistance that either of the original parts.  This third plasmid is called the construction plasmid and will always be digested with EcoRI and PstI. 
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===To place part A upstream of part B===
 +
*Part A - EcoRI & SpeI (insert)
 +
*Part B - XbaI & PstI  (vector)
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===To place part A downstream of part B===
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*Part A - XbaI & PstI  (insert)
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*Part B - EcoRI & SpeI (vector)
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E= EcoRI
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E= EcoRI<br>
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X= XbaI
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X= XbaI<br>
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S= SpeI
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S= SpeI<br>
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P= PstI
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P= PstI<br>
==Notes==  
==Notes==  
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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.
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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 [[Synthetic Biology:BioBricks/3A assembly|3A assembly]] or [[Amplified Insert Assembly]].

Current revision

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-----

Amplified Insert Assembly & Standard Assembly Digests

Both amplified insert assembly and standard assembly operate on the distinction between the vector part (the part which will stay in its plasmid) and the insert part (the part which will lave its plasmid and be placed in the vector plasmid next to the vector part). With these methods either part A or part B could be the vector or insert. If you're performing Standard Assembly there is an advantage to making the larger part the insert because it will be easier to gel-extract. If you're performing Amplified Insert Assembly there is an advantage to making the smaller part the insert because the PCR amplification step will go faster. For our example we'll choose part A as the insert part.

To place part A upstream of part B

  • Part A - EcoRI & SpeI (insert)
  • Part B - EcoRI & XbaI (vector)

To place part A downstream of part B

  • Part A - XbaI & PstI (insert)
  • Part B - SpeI & PstI (vector)

Three-Antibiotic Assembly Digests

In 3A assembly both part A and part B will be inserted into a third plasmid with a different antibiotic resistance that either of the original parts. This third plasmid is called the construction plasmid and will always be digested with EcoRI and PstI.

To place part A upstream of part B

  • Part A - EcoRI & SpeI (insert)
  • Part B - XbaI & PstI (vector)

To place part A downstream of part B

  • Part A - XbaI & PstI (insert)
  • Part B - EcoRI & SpeI (vector)


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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