Richard Lab:Amplified insert assembly

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Back to [[Richard_Lab:protocols | Protocols]]
Back to [[Richard_Lab:protocols | Protocols]]
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<center>'''NEW STANDARD ASSEMBLY'''</center>
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<center>'''Amplified Insert Assembly'''</center>
==Overview==
==Overview==
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This is a lab specific protocol.  For more information on the benefits of Amplified Insert Assembly see the [[Amplified Insert Assembly|consensus protocol]].  A You-Tube [http://www.youtube.ug/watch?v=Tvxx2bL0R2I&context=C3df62b2ADOEgsToPDskIOrrbFVhSy4joiAlQXxIYp video summary of the Amplified Insert Assembly method] provides a quick and entertaining visual introduction.
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This is what [[User:Michael A. Speer|Mike]] calls "New Standard Assembly" and is a method of "bio-bricking" two biological parts (i.e. pieces of DNA) together.  For more information on bio-bricking see [http://partsregistry.org/Help:Contents this link].  This method combines the ease and speed of 3A assembly with the reliability of standard assembly.  Major benefits of this assembly method over other assembly methods include:
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*no need for [[Richard Lab:Agarose Gel Electrophoresis|gel electrophoresis]] or [[DNA Gel extraction|gel extraction]].
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*the ability to insert small (i.e. invisible on a gel) parts.
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*no need to fool with [[Synthetic Biology:BioBricks/3A assembly|multiple antibiotic resistances]].
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*no having to make construction vectors.
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*really low background transformation
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This protocol is typically used to do bio-brick assembly with restriction sites consisting of the following configuration:
This protocol is typically used to do bio-brick assembly with restriction sites consisting of the following configuration:
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The two parts you want to assemble will be labeled "insert" and "vector" and will be initially contained on separate plasmids.  The eventual goal of assembly is to get these parts on the same plasmid next to one another.
The two parts you want to assemble will be labeled "insert" and "vector" and will be initially contained on separate plasmids.  The eventual goal of assembly is to get these parts on the same plasmid next to one another.
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==Materials==
 
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===General===
 
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*Pipettors
 
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*Microcentrifuge
 
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*Water baths
 
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*Thermocycler
 
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*Electroporator
 
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*Selective media plates
 
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===Enzymes===
 
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*EcoRI Restriction Endonuclease
 
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*XbaI Restriction Endonuclease
 
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*SpeI Restriction Endonuclease
 
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*PstI Restriction Endonuclease
 
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*[http://www.neb.com/nebecomm/products/productR0176.asp DpnI] Restriction Endonuclease
 
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*[http://www.neb.com/nebecomm/products/productM0254.asp Vent] DNA Polymerase (or another equivalent high fidelity polymerase)
 
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*[http://www.neb.com/nebecomm/products/productM0289.asp Antarctic Phosphatase]
 
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*T4 Ligase
 
==Procedure==
==Procedure==
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#[[miniprep|Miniprep]] both "insert" and "vector" from their respective cultures using [http://www.omegabiotek.com/products.php?CateID=11 kit] or [[Miniprep/GET buffer|this protocol]] (30 mins).
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1.  [[miniprep|Miniprep]] both "insert" and "vector" from their respective cultures using a [http://www.omegabiotek.com/products.php?CateID=11 kit] or [[Miniprep/GET buffer|this protocol]] (30 mins).<br>
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#PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away).  
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2.  PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away while the insert PCR is cycling).<br>
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::*Use a high-fidelity polymerase (e.g. pfu Turbo or Vent).
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::*Use a high-fidelity polymerase (e.g. pfu Turbo or Vent).<br>
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::*Use the same primers you use for colony PCR (Annealing Temp of 55°C).
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::*Use the same primers you use for colony PCR (Annealing Temp of 55-60°C).<br>
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#[[Richard Lab:Restriction Digest|Digest]] the "vector" for 2 hours.
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::*Only run 25-30 cycles as this will help ensure high fidelity.<br>
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#Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids|this protocol]].
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3.  Start [[Richard Lab:Restriction Digest|Digesting]] the "vector" while the PCR is cycling.<br>
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#[[Richard Lab:Restriction Digest|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).
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::*For help on deciding which enzymes to use see [[Enzyme selection for BioBricks digest|this page]].
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#Add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done.
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4.  Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids|this protocol]].<br>
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#Kill all reactions by incubating for 20 mins at 80°C.
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5.  [[Richard Lab:Restriction Digest|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).<br>
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#[[Richard Lab:Ligation|Ligate]] (Don't forget to kill the ligation).
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6.  Once the insert is digesting add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done.<br>
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#[[Richard Lab:Electroporation of E. coli|Transform]].
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7.  Kill all reactions by incubating for 20 mins at 80°C.<br>
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#Plate on plates with the same antibiotic as the "vector" resistance.
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8.  [[Richard Lab:Ligation|Ligate]] at a molar ratio of 4:1 (insert:vector).<br>
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#Celebrate.
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9.  [[Richard Lab:Electroporation of E. coli|Transform]].<br>
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10. Plate on plates with the same antibiotic as the "vector" resistance.<br>
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11. Celebrate.
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*This entire process should take around 6 hours as many steps overlap. 
 
*If you already have PCR insert ready to go (i.e. you ran the PCR the night before from old miniprep) then it only takes about 4 hours.
*If you already have PCR insert ready to go (i.e. you ran the PCR the night before from old miniprep) then it only takes about 4 hours.
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*The "insert" will only be digested for one hour.  This is okay as there is a lot of it.
*The "insert" will only be digested for one hour.  This is okay as there is a lot of it.
*Detractors of this method may say that it's risky to PCR the inserts because of mutations.  We say:
*Detractors of this method may say that it's risky to PCR the inserts because of mutations.  We say:
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**This hasn't been a problem for us.
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::#This hasn't been a problem for us.
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**This is why we use a high-fidelity polymerase
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;;#This is why we use a high-fidelity polymerase
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**We're sequencing the constructs anyway so we'd spot any mutations.
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::#We're sequencing the constructs anyway so we'd spot any mutations.
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==References==
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*Speer, M.A. and T.L. Richard.  2011.  [http://www.jbioleng.org/content/5/1/17 '''Amplified insert assembly: an optimized approach to standard assembly of BioBrick™ genetic circuits''' ''Journal of Biological Engineering'' 5:17]
==Contact==
==Contact==
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*[[user:Michael A. Speer | Mike]]
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*This protocol was developed by [[user:Michael A. Speer | Mike Speer]] and [http://www.abe.psu.edu/fac/Richard/Overview.htm Dr. Tom Richard]
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or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
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*You can also discuss this protocol [[Talk:{{PAGENAME}}|here]].  

Current revision

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Amplified Insert Assembly

Contents

Overview

This is a lab specific protocol. For more information on the benefits of Amplified Insert Assembly see the consensus protocol. A You-Tube video summary of the Amplified Insert Assembly method provides a quick and entertaining visual introduction.

This protocol is typically used to do bio-brick assembly with restriction sites consisting of the following configuration:

-----EcoRI--XbaI--Part--SpeI--PstI-----

Ocassionally other enzymes (e.g. BamHI or HindIII) are used to make protein fusions. See our bio-brick format page for more details.

The two parts you want to assemble will be labeled "insert" and "vector" and will be initially contained on separate plasmids. The eventual goal of assembly is to get these parts on the same plasmid next to one another.

Procedure

1. Miniprep both "insert" and "vector" from their respective cultures using a kit or this protocol (30 mins).
2. PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away while the insert PCR is cycling).

  • Use a high-fidelity polymerase (e.g. pfu Turbo or Vent).
  • Use the same primers you use for colony PCR (Annealing Temp of 55-60°C).
  • Only run 25-30 cycles as this will help ensure high fidelity.

3. Start Digesting the "vector" while the PCR is cycling.

  • For help on deciding which enzymes to use see this page.

4. Purify the PCR product using a kit or this protocol.
5. Digest insert for 1 hour (adding DpnI along with the other restriction endonucleases).
6. Once the insert is digesting add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done.
7. Kill all reactions by incubating for 20 mins at 80°C.
8. Ligate at a molar ratio of 4:1 (insert:vector).
9. Transform.
10. Plate on plates with the same antibiotic as the "vector" resistance.
11. Celebrate.

  • If you already have PCR insert ready to go (i.e. you ran the PCR the night before from old miniprep) then it only takes about 4 hours.

Notes

  • The DpnI eliminates any background from the insert PCR.
  • The phosphatase eliminates any background vector.
  • The "vector" will be digested for a total of thee hours (including nearly one hour with Antarctic Phosphatase)
  • The "insert" will only be digested for one hour. This is okay as there is a lot of it.
  • Detractors of this method may say that it's risky to PCR the inserts because of mutations. We say:
  1. This hasn't been a problem for us.
  2. This is why we use a high-fidelity polymerase
  3. We're sequencing the constructs anyway so we'd spot any mutations.

References

Contact

Back to Protocols

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