Site-Directed Mutagenesis of Plasmids

We frequently use both the Phusion system from New England Biolabs and the Site-Directed Mutagenesis Kits from Stratagene for site-directed mutagenesis. Below are examples of each procedure.

Using Site-Directed Mutagenesis to Install BsaI sites into a plasmid for subsequent use in Golden Gate Cloning
Authors: Eldon Chou, Caroline Ajo-Franklin, contributions from: Guillaume Cambray
Overview:This procedure describes the first part of making a promoter library in a given plasmid. Specifically this protocol describes using the Phusion site-directed mutagenesis kit to install BsaI sites flanking the promoter in the plasmid. To complete the library, Golden Gate cloning is used with dsDNA oligos to generate plasmids with different promoters, which is described in a separate protocol.

Materials & Equipment needed:

  • parent plasmid from which to create variants, i.e. pEC86
  • 10x T4 Ligase buffer, NEB # B0202S
  • T4 Polynucleotide kinase, 10 U/uL NEB # M0201S
  • Phusion Site-Directed High G-C Mutagenesis kit, # M0531S (or 5x High G-C buffer, dNTPs, DMSO, & Hot Start Phusion DNA Polymerase)
  • DpnI, NEB # R0176S
  • NEB Buffer 4 (comes with DpnI)
  • BsaI HF, NEB # R3535S
  • PCR machine

Critical Steps

  1. Check your plasmid to ensure it contains no BsaI sites before beginning this procedure.

Important Safety Concerns:
Waste Disposal & Clean-up:
Procedure:
Day 0: Design mutagenesis primers

  1. Design primers.

Day 1: Phosphorylate mutagenesis primers and do pcr mutagenesis of plasmid
Note: The first step (phosphorylation) is unnecessary if you ordered phosphorylated primers.

    1. Set-up reactions for each primer separately.For each reaction, mix in a 200uL pcr tube:
      • 2 uL 10x T4 Ligase buffer
      • 2 uL 100 uM primer in ddH2O
      • 0.5 uL T4 polynucleotide kinase (PNK; 1U/uL)
      • 15.5 uL ddH20
      • total volume = 20 uL
    2. Using a pcr machine, heat at 37°C for 2 hours to phosphorylate the primers.
    3. Then heat at 95°C for 5 minutes to heat inactivate the PNK enzyme.
    4. Set-up iPCR. For each PCR reaction, mix:
      • 0.25 uL template (plasmid)
      • 0.5 uL 1 uM phosphorylated forward primer (from step 1)
      • 0.5 uL 1 uM phosphorylated reverse primer (from step 1)
      • 10 uL  5x High G-C buffer
      • 1 uL dNTP
      • 2 uL DMSO
      • 0.5 uL Hot Start Phusion DNA Polymerase
      • 35.25 uL ddH2O

      total volume= 50 uL

    5. Run iPCR with the following steps:

Temperature

Time (s)

1.  Activate

95°C

300s

2.  Melt

95°C

8s

3.  Anneal

60°C

240s

4.  Elongate

72°C

30s

5.  Cycle 25 times

Go to Step 2

25 times

6.  Hold

4°C

Hold
  1. Digest template (plasmid) DNA using DpnI. DpnI will cut methylated DNA (i.e. DNA produced inside a bacteria) and will leave un-methylated (i.e. pcr-generated DNA) intact.For each iPCR reaction, mix:
  • 25 uL PCR reaction
    • 3 uL 10x NEB Buffer 4
    • 1uL water
    • 1uL DpnI
    • Total volume = 30 uL
  • Gel purify linear pcr products.

Day 2: Ligation of plasmid and Transformation
Ligate pcr products into circular plasmids using T4 DNA Ligase. Cells will typically eliminate linearized plasmids, but will propagate circularized plasmids.

  1. Mix:
    • 5 uL gel purified linear pcr product
    • 1 uL 10x T4 DNA Ligase buffer
    • 3 uL ddH2O
    • 1 uL T4 DNA Ligase
    • Total = 10 uL
  2. Transform pcr products into chemically competent cells.
  3. Incubate 200μL of chemically competent Mach 1’s with 200ng plasmid for 30 minutes on ice.
  4. Heat shock at 42°C for 45 seconds.
  5. Incubate on ice for 2 minutes.
  6. Shake / incubate at 30°C for 1 hour.
  7. Spin down at 4krpm for 5 minutes, decant, resuspend in 100μL and plate on a plate with chloramphenicol.

Days 3-5. See Plasmid Verification.Then Move onto Golden Gate cloning.

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