Richard Lab:Amplified insert assembly

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*SpeI Restriction Endonuclease
*SpeI Restriction Endonuclease
*PstI Restriction Endonuclease
*PstI Restriction Endonuclease
-
*DpnI Restriction Endonuclease
+
*[http://www.neb.com/nebecomm/products/productR0176.asp DpnI] Restriction Endonuclease
-
*Vent DNA Polymerase
+
*[http://www.neb.com/nebecomm/products/productM0254.asp Vent] DNA Polymerase (or another equivalent high fidelity polymerase)
-
*Antarctic Phosphatase
+
*[http://www.neb.com/nebecomm/products/productM0289.asp Antarctic Phosphatase]
*T4 Ligase  
*T4 Ligase  
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#[[miniprep|Miniprep]] both "insert" and "vector" from their respective cultures using [[Miniprep/Qiagen kit|a kit]] or [[Miniprep/GET buffer|this protocol]] (30 mins).
#[[miniprep|Miniprep]] both "insert" and "vector" from their respective cultures using [[Miniprep/Qiagen kit|a kit]] or [[Miniprep/GET buffer|this protocol]] (30 mins).
#PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away).  
#PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away).  
-
::*Use a high-fidelity polymerase (e.g. pfu Turbo or [http://www.neb.com/nebecomm/products/productM0254.asp Vent]).
+
::*Use a high-fidelity polymerase (e.g. pfu Turbo or Vent).
::*Use the same primers you use for colony PCR (Annealing Temp of 55°C).
::*Use the same primers you use for colony PCR (Annealing Temp of 55°C).
#[[Richard Lab:Restriction Digest|Digest]] the "vector" for 2 hours.
#[[Richard Lab:Restriction Digest|Digest]] the "vector" for 2 hours.
#Purify the PCR product using a kit or [[Ethanol precipitation of nucleic acids|this protocol]].
#Purify the PCR product using a kit or [[Ethanol precipitation of nucleic acids|this protocol]].
-
#[[Richard Lab:Restriction Digest|Digest]] insert for 1 hour (adding [http://www.neb.com/nebecomm/products/productR0176.asp DpnI] along with the other restriction endonucleases).
+
#[[Richard Lab:Restriction Digest|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).
#Add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done.
#Add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done.
#Kill all reactions by incubating for 20 mins at 80°C.
#Kill all reactions by incubating for 20 mins at 80°C.
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**This is why we use a high-fidelity polymerase
**This is why we use a high-fidelity polymerase
**We're sequencing the constructs anyway so we'd spot any mutations.
**We're sequencing the constructs anyway so we'd spot any mutations.
-
 
-
==References==
 
==Contact==
==Contact==

Revision as of 18:20, 25 January 2011

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NEW STANDARD ASSEMBLY

Contents

Overview

This is what 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 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:

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.

Materials

General

  • Pipettors
  • Microcentrifuge
  • Water baths
  • Thermocycler
  • Electroporator
  • Selective media plates

Enzymes

  • EcoRI Restriction Endonuclease
  • XbaI Restriction Endonuclease
  • SpeI Restriction Endonuclease
  • PstI Restriction Endonuclease
  • DpnI Restriction Endonuclease
  • Vent DNA Polymerase (or another equivalent high fidelity polymerase)
  • Antarctic Phosphatase
  • T4 Ligase

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).
  • Use a high-fidelity polymerase (e.g. pfu Turbo or Vent).
  • Use the same primers you use for colony PCR (Annealing Temp of 55°C).
  1. Digest the "vector" for 2 hours.
  2. Purify the PCR product using a kit or this protocol.
  3. Digest insert for 1 hour (adding DpnI along with the other restriction endonucleases).
  4. Add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done.
  5. Kill all reactions by incubating for 20 mins at 80°C.
  6. Ligate (Don't forget to kill the ligation).
  7. Transform.
  8. Plate on plates with the same antibiotic as the "vector" resistance.
  9. Celebrate.

This entire process should take around 6 hours as many steps overlap. If you already have PCR insert ready to go 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:
    • This hasn't been a problem for us.
    • This is why we use a high-fidelity polymerase
    • We're sequencing the constructs anyway so we'd spot any mutations.

Contact

or instead, discuss this protocol.

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