NanoBio: Knockout/in

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(New page: ==Knockout/in== For an example of this, see Heather's notebook C028 (notebook C pg28) The best way to do a knockin is to make the linear fragment of DNA for integration into the genome w...)
(Knockout/in)
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==Knockout/in==
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==Introduction==
For an example of this, see Heather's notebook C028 (notebook C pg28)
For an example of this, see Heather's notebook C028 (notebook C pg28)
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The best way to do a knockin is to make the linear fragment of DNA for integration into the genome with SOE pcr. After this fragment is made and verified by sequencing, you may follow the lambda red protocol as with a normal gene knockout.
The best way to do a knockin is to make the linear fragment of DNA for integration into the genome with SOE pcr. After this fragment is made and verified by sequencing, you may follow the lambda red protocol as with a normal gene knockout.
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Check out a good summary of SOE pcr at bch.msu.edu ([http://www.bch.msu.edu/faculty/kroos/supplement.htm])
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Check out a good summary of SOE pcr at bch.msu.edu ([http://www.bch.msu.edu/faculty/kroos/supplement.htm]) This method is also used in site directed mutagenesis. (See image below)
[[Image:SOE PCR.jpg]]
[[Image:SOE PCR.jpg]]
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#PCR3b: use PCR3a as the primer and the extreme ends of the desired product as the primer. run 35 rounds.
#PCR3b: use PCR3a as the primer and the extreme ends of the desired product as the primer. run 35 rounds.
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This protocol will describe how you design the 6 primers necessary:
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==Protocol==
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#
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===Primer Design===
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#First draw the product you'd like to make:
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H1 - GeneIn -P1 - FRT - AbR - FRT - P2 - H2
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#*The H1 and H2 will designate where your fragment will insert into the genome.
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#*P1 and P2 are the same as in the original protocol
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#*P1 will be used as the overlap region
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#*The antibiotic resistance gene will depend on which plasmid template you use.
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#**pKD3 = ChlR
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#**pKD4 = KanR
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#*
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===Protocol===

Revision as of 15:03, 10 August 2009

Contents

Introduction

For an example of this, see Heather's notebook C028 (notebook C pg28)

The best way to do a knockin is to make the linear fragment of DNA for integration into the genome with SOE pcr. After this fragment is made and verified by sequencing, you may follow the lambda red protocol as with a normal gene knockout.

Check out a good summary of SOE pcr at bch.msu.edu ([1]) This method is also used in site directed mutagenesis. (See image below)

Image:SOE PCR.jpg The general procedure is:

  1. PCR1: 5' half of the desired fragment. the last 20-30bp of the 3' end should complement the 5' end of PCR2
  2. PCR2: 3' half of the desired fragment. the first 20-30bp of the 5' end should complement the last basepairs of PCR1
  3. PCR3a: use PCR1 and PCR2 as both the template and the primer. run 20 rounds of PCR this way
  4. PCR3b: use PCR3a as the primer and the extreme ends of the desired product as the primer. run 35 rounds.

Protocol

Primer Design

  1. First draw the product you'd like to make:
H1 - GeneIn -P1 - FRT - AbR - FRT - P2 - H2
    • The H1 and H2 will designate where your fragment will insert into the genome.
    • P1 and P2 are the same as in the original protocol
    • P1 will be used as the overlap region
    • The antibiotic resistance gene will depend on which plasmid template you use.
      • pKD3 = ChlR
      • pKD4 = KanR

Protocol

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