NanoBio: Knockout/in

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)

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

First draw the product you'd like to make:

     H1 - GeneIn - P1 - FRT - AbR - FRT - P2 - H2

1. The H1 and H2 will designate where your fragment will insert into the genome.
2. P1 and P2 are the same as in the original protocol
3. P1 will be used as the overlap region
4. The antibiotic resistance gene will depend on which plasmid template you use.
   • pKD3 = ChlR
   • pKD4 = KanR

Break apart your desired fragment into the pieces you can make from what is available:

     H1 - GeneIn - P1                                 = PCR1
                   P1 - FRT - AbR - FRT - P2 - H2     = PCR2

1. Primers for PCR1
   1. Design "regular" primers for the gene you'd like to incorporate (see: )
   2. On the 5' end of the upstream primer, add an H1 appendage.
      • H1-InA
   3. On the 3' end of the downstream primer, add a P1 appendage. Make sure to reverse complement the entire primer as a whole.
      • InB-P1....order the reverse complement [InB-P1]rc
2. Primers for PCR2
   1. The upstream primer is P1, no appendage
   2. The downstream primer is P2-H2. Make sure to reverse complement the entire primer. [P2-H2]rc
3. Primers for PCR3b
   1. These are just the extremes of the fragment. So the 1st 25 bases of H1 and the last (rc) 25 bases of H2.

Protocol

1. Dissolve primers to 100uM (10xnmol uL of water)
2. Do PCR1 and PCR2 using Pfx dna polymerase as described here:
   1. PCR1:
      • Template = origin of gene to be knocked in
      • Ta = 55°C
      • Extension time = #bp/1000 minutes
   2. PCR2:
      • Template = either pKD3(ChlR) or pKD4(KanR)
      • Ta = 55°C
      • Extension time = 2 minutes for both
   3. Make master mix:..............μl/rxn
      1. 10x PCR buffer........25.0
      2. 10mM dNTPs............3.0
      3. 50mM Mg₂SO₄.........2.0
      4. 10x enhancer..........30.0
      5. ddH₂O....................34.0
      • Note: mix together {n+1} volumes of each substrate, where n=the number of reactions you will be doing.
      • Note: these volumes are for 100uL reactions. Adjust if using lower volumes.
   4. Make 10μM primer mix:
      1. Mix 2μL of both primers (100μM stock) into 16μL ddH₂O.
      2. If you need more than 20μL of primer, adjust volumes. (1μL of each per total 10μL mix)
   5. For each reaction, mix together in PCR tube.
      1. 94μL Master Mix
      2. 1μL Template DNA
      3. 3μL Primer mix (10μM each primer)
      4. 2μL Pfx Platinum DNA Pol
3. PCR purify
4. Measure concentrations and run on a gel to confirm length.
5. PCR3a
   1. Template/Primer: PCR1+PCR2. Use ~500ng of each in there.
   2. Ta = 55°C
   3. Extension time = #bp/1000 minutes.
   4. Run for 20 cycles
   5. Run a single PCR tube:..............μl/rxn
      1. 10x PCR buffer........12.5
      2. 10mM dNTPs............1.5
      3. 50mM Mg₂SO₄.........1.0
      4. 10x enhancer..........15.0
      5. ddH₂O....................Adjust depending on concentration of PCR1/2
      6. ____μL PCR1 (~500ng)
      7. ____μL PCR2 (~500ng)
      8. 1μL Pfx Platinum DNA Pol
      • Note: these volumes are for 50uL reactions.

1. PCR3b
   2. Make master mix:..............μl/rxn
      1. 10x PCR buffer........25.0
      2. 10mM dNTPs............3.0
      3. 50mM Mg₂SO₄.........2.0
      4. 10x enhancer..........30.0
      5. ddH₂O....................34.0
      • Note: mix together {n+1} volumes of each substrate, where n=the number of reactions you will be doing.
      • Note: these volumes are for 100uL reactions. Adjust if using lower volumes.
   3. Make 10μM primer mix:
      1. Mix 2μL of both primers (100μM stock) into 16μL ddH₂O.
      2. If you need more than 20μL of primer, adjust volumes. (1μL of each per total 10μL mix)
   4. For each reaction, mix together in PCR tube.
      1. 94μL Master Mix
      2. 1μL Template DNA
      3. 3μL Primer mix (10μM each primer)
      4. 2μL Pfx Platinum DNA Pol

Return to Protocols.