Richard Lab:Site Directed Mutagenesis

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Introduction

These protocols are used if you want to change one to three consecutive bases in a sequence (like removing a restriction site or changing an amino acid). They both utilize a fast high-fidelity non-displacing DNA polymerase (Phusion) to replicate the plasmid including the desired mutation. DpnI then cuts up all your old plasmid. While these protocols can make reliable controlled mutations, the constraints may not work for you; check out the consensus protocol for more options. Also please read both protocols to see which one you want to try.

Double Primer Method

This protocol uses two oligos, each containing the desired mutation. It is more reliable than the other protocol, but can only mutate at one location (1-3bp) at a time.

Method

1. Design mutagenesis primers.

  • The targeted mutation should be included into both primers.
  • The mutation can be as close as 4 bases from the 5-terminus.
  • The mutation should be at least 8 bases from the 3-terminus.
  • At least eight non-overlapping bases should be introduced at the 3-end of each primer.
  • At least one G or C should be at the end of each primer.
  • Design your primers (including the mutations) to have a Tm >=78°C.

2. Purify template plasmid from a dam+ E. coli strain via miniprep.
3. Set up mutagenesis PCR mix:

  • 36µl water
  • 10µl 5X Phusion Buffer
  • 1µl dNTPs (25mM each)
  • 1µl Primer F
  • 1µl Primer R
  • 0.5µl Template DNA
  • 0.5µl Phusion Polymerase

4. Run PCR

  1. 98°C for 30 secs
  2. Run the following for 20 cycles:
    1. 98°C for 10 secs
    2. 60°C for 30 secs
    3. 72°C for 30 sec/kb of plasmid length minimum
  3. 72°C for 5 mins
  4. 4°C infinite

5. Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary)
6. Incubate 2-3 hours at 37°C.
7. Purify PCR product and elute into 30μL.
8. Transform 3μL purified DNA into highly competent cells.
9. Screen the transformants for the desired mutation using restriction digest or sequencing.

Notes

  • In Mike's capable hands 70% of colonies are correct using this protocol
  • Just pick two colonies to sequence and at least one of them will have the mutation.
  • We like to order oligos from IDT. They have a great turn-around time, can do oligos up to 60bp, always do a good job, and are cheap.

references

Zheng, L., U. Baumann, and Jean-Louis Reymond. 2004. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 2004; 32(14): e115.

Single Primer Method

This protocol only uses one oligo per mutation site (cuts the oligo cost in half) and can mutate more than one site at a time. While this protocol is less reliable than the previous protocol (only ~25% of colonies will contain the desired mutation(s)), if you're doing multiple mutations on the same piece of DNA than this can save you many days of work by just sequencing a few more colonies. In this protocol there is an initial Kinase (phosphorylation) reaction to allow your primers to be available for ligation. The polymerase will extend from one primer to another and then the Taq ligase will seal the nick. This allows multiple mutations to be done at the same time.

Method

1. Design mutagenesis primer(s).

  • The targeted mutation should be in the middle of the primer
  • Design your primers (including the mutations) to have a Tm >=78°C.

2. Purify template plasmid from a dam+ E. coli strain via miniprep.
3. Set up mutagenesis PCR mix:

  • Water
  • Phusion HF Buffer
  • Ligase Buffer
  • Primers
  • dNTPs
  • Template
  • PNK
  • Taq Ligase
  • Phusion Polymerase (hotstart)

4. Run PCR

  1. 37°C for 30 minutes (this is the kinase step)
  2. 98°C for 30 secs
  3. Run the following for 20 cycles:
    1. 98°C for 10 secs
    2. 60°C for 30 secs
    3. 72°C for 30 sec/kb of plasmid length minimum
  4. 72°C for 5 mins
  5. 4°C infinite

5. Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary)
6. Incubate 4-6 hours at 37°C.
7. Purify PCR product and elute into 30μL.
8. Transform 3μL purified DNA into highly competent cells.
9. Screen the transformants for the desired mutation using restriction digest or sequencing.

Notes

  • Because the inital kinase step could lead to non-specific binding, it would be best to use a hot-start polymerase for this procedure.

References

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