# Troublesome Site-Directed Mutagenesis: Troubleshooting Your Experiment for Stubborn Mutations

As is sadly the case in many experiments, site-directed mutagenesis (SDM) does not always work the way we would like it to the first time around. Let’s discuss some of the comment problems, and the site-directed mutagenesis tips for overcoming them.

Some common problems include (but are not limited to):

• Getting too many colonies.
• Not getting any colonies.
• Getting colonies that have not incorporated the desired mutation successfully.

The most important site-directed mutagenesis tip I can offer here is to always, always, always run control reactions alongside your SDM reactions! Many kits come with the components to run control reactions side by side with your experimental ones. Use of these controls will help you narrow down where exactly your protocol is not working and will save you time and reagents in the long run.

Here are a few site-directed mutagenesis tips to help you on your way when trying to troubleshoot a bothersome reaction!

## Site-Directed Mutagenesis Tips for Troubleshooting

### When You Get Too Many Colonies

• Decrease the concentration of template DNA used in the PCR reaction.
• Decrease the amount of PCR product you are using in the transformation.
• Plate several concentrations of your transformed suspension (e.g. 10 µl of bacterial prep, 20 µl, 50 µl, 100 µl) and only pick colonies from a plate with well-spaced colonies.
• Increase the DpnI digestion time (e.g. 2 h instead of 1 h).

### When You Get No Colonies

• Increase the amount of template DNA used in the PCR reaction.
• Increase the amount of PCR product you are using in the transformation.
• Try a temperature gradient – many PCR machines can be set up to run a variety of temperatures across the block during the program. Start by trying 3 to 4 different temperatures and optimize from there.
• Try altering the extension temperature or time, e.g. drop extension temperature to 68°C and extend at 60 seconds/kb.
• Add a little DMSO (2-8%) to disrupt base pairing and assist in strand separation in GC rich regions.
• Check that your competent cells are working with a control transformation.
• Ethanol precipitate the digested DNA and resuspend in a smaller volume before transformation.
• Clean up your DNA sample to remove salts and other substances leftover from the PCR reaction before transformation.
• Increase the concentration of MgCl2.

### Colonies Forming Without the Desired Mutation

• Use E. coli host bearing dam-methylase for the template plasmid preparation, such as JM109 or DH5$\alpha$.
• Increase the DpnI digestion time (e.g. 2 h instead of 1 h) or increase the amount of DpnI used (but this may add to the expense of your experiment!).
• Plate several concentrations of your transformed suspension (e.g. 10 µl of bacterial prep, 20 µl, 50 µl, 100 µl) and only pick colonies from a plate with well-spaced colonies.
• Decrease the number of PCR cycles.

### Still Nothing?

Redesign your primers and try again!

As we mentioned previously, site-directed mutagenesis primers should typically be around 30 bp long with the mutated site as close to the center as possible with roughly 12 bp on either side.

### Make the least number of changes possible

For example, to change serine to alanine starting with a UCA codon, change it to GCA (1 change) instead of GCU (2 changes).

### Aim for Symmetry

Keep your region of interest as close to the middle of the primer as possible.

### Keep GC Content at Approximately 50%

Some regions are easier to work with than others for this rule, when in doubt aim for higher rather than lower GC content as you can adjust your PCR temperature setting to account for the changes in annealing/denaturing temperature that higher GC content will have.

### Start and Finish Your Primer with Either Matching Gs or Cs

G and C bases bind with higher affinity than T or A, so starting and finishing your primer with GG or CC will help with the initial binding. If you can’t manage GG at both ends, try for GG at one end and G at the other.

Be careful: Don’t start with GG and finish with CC – you’ll end up with self-annealing primers!

### Mutating Multiple Sites?

If the sites are close together consider using overlapping primers or partially overlapping primers, but make sure the sites are mutated in both sets of primers.

What are your site-directed mutagenesis tips for troubleshooting a tough mutation? Let us know in the comments below!

Originally published July 9, 2016. Reviewed and updated on November 27, 2020.

1. Jessica on March 27, 2017 at 2:08 pm

Hi, I have been trying unsuccessfully to make a simple two nucleotide mutation. I checked that my primers worked and I do get colonies. The issue is when I colony check with the outside plasmid primers, I get multiple bands. I have sent this in for sequencing and it comes back a mess. I am looking for a band around 700 bp, which I get, but I also get a band around 2000 bp and 500 bp. I’m not sure what’s going on here and would really like to move on.

2. komalshamnani on February 3, 2017 at 7:36 am

Hello! I have been trying site directed mutagenesis. In my case some random muations are occuring along with desired one and eventually I dont get expression of desired protein. i have to do only one nucleotide mutation to change one amino acid. can some one suggest me what should i do?

• Bego on April 12, 2017 at 7:03 pm

Hello Komalshamnani

I am experiencing exactly the same problem as you. I get PCR product of expected size and colonies, and when sent to sequence the mutation is present. However other deletions are present in the same frameshift which is probably why my protein expression fails. I have red to redesign primers that overlap, but I’m not sure if this would work.
Did you figure out how to fix your problem? I would really appreciate some help if you managed to troubleshoot this.

Thanks!

3. K Clarke on November 12, 2016 at 3:04 am

Hello,
I had to do some site-directed mutagenesis (~6 separate mutations) and ended up using partially overlapping primers rather than Qiagens recommendation for large complementary primers, and it worked quite well. It may be a worthwhile alternative to investigate for anyone that has continued problems with SDM. Linked below is a paper describing the system as well as a kit protocol that could be ordered or easily home brewed.

4. Matt Hoss on December 7, 2015 at 2:03 pm

Great tips. I usually like my primers closer to 45bp so I can 2-step the PCR. Few hairpins make it at 72.

5. GK Adam on December 7, 2015 at 1:05 pm

I use Agilent’s Quick Change primer design tool (http://www.genomics.agilent.com/primerDesignProgram.jsp). Its very easy to use and has never failed me in 5+ years. I would highly recommend using it for SDM primer design.

• Haoran Yu on December 8, 2015 at 4:05 pm

Totally agree with you

• J Ch on September 28, 2016 at 6:36 pm

Hi, I am having problems with the Agilent QuikChange II kit. I have tried trice and neither resulted in any colonies. When I run a gel using 5ul of the PCR reaction, I do not see any bands. I used the design program to design my primers and they are 31bp long with a single base change to T->A at the center, and they end with G/Cs. Here is my protocol:
5ul reaction buffer
1ul dNTPs
10ng template DNA(0.4ul of 500ng/ul stock that I diluted 1:20)
125ng primers(0.5ul of 1mg/ml stock that I diluted 1:4-so 2ul of each primers with 4ul of water)
43.1ul water
I followed the manual’s cycling parameters with 12 cycles and ay 68C for 9:12(the template is 9175kb)
Add 1ul DpnI digest for 1hr at 37C
Transform into Invitrogen Max Efficiency Competent Cells.

I tried using Phusion instead of Pfu once, but also did not get any colonies. I also tried transf0rming more of the DpnI digested product, but also did not get any colonies.

• Dr Amanda Welch on September 28, 2016 at 7:11 pm

Have you done the control PCR? This will give you an idea if something else in the kit is not working.

The other thing to look at is that your template DNA is rather long (over 9kb). You might want to try subcloning in a smaller plasmid and then into your desired vector. Good luck!

• J Ch on September 28, 2016 at 7:21 pm

I have not tried the control. The kit if have is over a year old. I am not sure if that affects the efficiency substantially. But I will try on a new kit.

• J Ch on October 6, 2016 at 6:18 am

I tried the positive control and see bands on it when I run a gel but not on my mutagenesis product. I also tried adding 3 and 5%DMSO but still no product. Does anyone know what might be the problem?

• Dr Amanda Welch on October 6, 2016 at 1:22 pm

Have you tried adjusting your annealing temperature? Or adding MgCl2? If you have and it still doesn’t work, then it sounds like you’ll need to design new primers. See if you can add more Gs or Cs to the ends.

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