Hi everyone,

I performing a site directed mutagenesis on a wildtype plasmid and I have been having troubles generating the mutant plasmid I want. I am hopping to make a post here and see if anybody have troubleshooting suggestions I can try.

I am mutating a lysine to a arginine in my target sequence. Since the target sequence if AT rich (29% GC), I adopted the AT-rich SDM protocol described in this paper (doi: 10.7171/jbt.20-3103-003). I have put the detailed protocol below. After the Dpn1 digestion, I always ran a little samples on gel to make sure that there are some product from the reaction.

1. Design overlapping oligos with the mutation site located in the center of each oligo

2. Oligo design:

a. Complementary forward and reverse oligos with mutation in the middle

b. Must have 10 or more bases of correct sequence on either side of the mismatch/deletion/insertion region (could be up to 20)

c. Ends must be G or C

d. Correct sequence flanks should be at least 25% G/C

3. PCR Reaction (50ul, one reaction)

• DNA Template: 500ng (10ng/ul)
• FOR Primer: 0.6ul (6pm total)
• REV Primer: 0.6ul (6pm total)
• 2.5mM dNTPs: 5ul
• Q5 high fidelity polymerase: 1ul
• 5X Q5 reaction buffer: 10ul
• MQ H2O: QS to 50ul

4. PCR Thermocycling

a. Program 1: The melting step through the extension step was cycled for an additional 4 times (5 cycles in total)

• Polymerase activation: 98°C for 30 s
• Melting: 98°C for 30 s
• Step-down annealing: 65°C–55°C (-2°C/cycle) for 1.5 min
• Extension: 68°C at 1kb/min
• Final Extension: 68°C for 1.0 min

b. Program 2: The melting step through the extension step was cycled for an additional 14 times (15 cycles in total)

• Polymerase activation: 98°C for 30 s
• Melting: 98°C for 30 s
• Annealing: 65°C for 1.5 min
• Extension: 68°C at 1kb/min
• Final Extension: 68°C for 2.0 min

5. Dpn1 restriction enzyme digest (destroys unmutated template DNA)

Add 10 units (1uL) of DpnI to 50uL PCR product. Incubate the tube at 37˚ in heat block for 3hrs.

6. Transform PCR product into DH5@

7. Verify mutation via diagnostic digestion via diagnostic digestion and whole plasmid sequencing.

For the primers, I used overlapping primers (31bps, Tm=63 degrees based on NEB) with the mutagenesis site in the middle (only one bp is modified). I double checked and made sure that primers doesn't have any trouble with hairpins or dimers.

By now I have performed the mutagenesis twice and sequenced 6 candidates. However, all the candidates seems to only contain the WT gene instead of the mutated gene. Therefore, I am hopping to see if I can get some help troubleshooting my site directed mutagenesis. Thanks in advance!