Richard Lab:Amplified insert assembly: Difference between revisions

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#Miniprep both "insert" and "vector" from their respective cultures (30 mins).
#Miniprep both "insert" and "vector" from their respective cultures (30 mins).
#PCR the insert plasmid (2 hrs)  
#PCR the insert plasmid (2 hrs)  
##Use a high-fidelity polymerase (eg. pfu Turbo or Deep Vent)
##Use a high-fidelity polymerase (eg. pfu Turbo or Deep Vent).
##Use the same primers you use for colony PCR.
##Use the same primers you use for colony PCR (Annealing Temp of 55°C).
#Begin Vector Digest.
#Digest vector for 2 hours.
#Purify the PCR product.
#Purify the PCR product.
#Begin Insert Digest (Adding DpnI).
#Digest insert for 1 hour (adding DpnI along with the other restriction endonucleases).
#Add DpnI restriction endonuclease to the "insert" digest.
#Add Antarctic Phosphatase to the "vector" digest and incubate until Insert Digest is done.
#Add Antarctic Phosphatase to the "vector" digest.
#Ligate with 3μL of vector and 3μL of insert in a 20ml Ligation.
#Ligate
#Transform.
#Transform
#Celebrate.
#Celebrate


==Notes==
==Notes==

Revision as of 15:56, 11 January 2011

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Overview

This is what Mike calls "Standard Assembly 2.0" 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 triple antibiotic assembly with the fidelity of standard assembly. Major benefits of this assembly method over other assembly methods include:

  • no gel electrophoresis or extractions
  • The ability to insert small (i.e. invisible on a gel) parts.
  • No need to fool with multiple antibiotic resistances.
  • No having to make construction vectors.

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.

Procedure

Your two parts will be thusly labeled "insert" and "vector" although initially they will both be contained on separate plasmids. The basic steps are as follows:

  1. Miniprep both "insert" and "vector" from their respective cultures (30 mins).
  2. PCR the insert plasmid (2 hrs)
    1. Use a high-fidelity polymerase (eg. pfu Turbo or Deep Vent).
    2. Use the same primers you use for colony PCR (Annealing Temp of 55°C).
  3. Digest vector for 2 hours.
  4. Purify the PCR product.
  5. Digest insert for 1 hour (adding DpnI along with the other restriction endonucleases).
  6. Add Antarctic Phosphatase to the "vector" digest and incubate until Insert Digest is done.
  7. Ligate with 3μL of vector and 3μL of insert in a 20ml Ligation.
  8. Transform.
  9. Celebrate.

Notes

References

Contact

or instead, discuss this protocol.

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