Protocols

1. Quick Protocol for Q5^® Site-Directed Mutagenesis Kit (Without Competent Cells) Quick (E0552)
2. Protocol for Q5^® Site-Directed Mutagenesis Kit (Without Competent Cells) (E0552)
3. Protocol for Control Reaction (E0552)

Manuals

Web Tools

• NEBaseChanger
• NEBcloner^®

FAQs

1. What is the maximum number of nucleotides that can be inserted with this kit?
2. What is the maximum distance that can be tolerated between substitutions?
3. Typically, what percentage of transformants will have the desired mutation incorporated?
4. What is the KLD Mix?
5. What types of competent cells are compatible with this kit?
6. If I double my PCR size, should I add more PCR mix to the KLD reaction?
7. Why is the desired mutation missing from the transformants that I screened?
8. Why do I not see my PCR product after using the Q5® Site-Directed Mutagenesis Kit?
9. What plasmid sizes can be amplified using the Q5® Site-Directed Mutagenesis Kit?
10. Do I need to purify my plasmid before or after the KLD reaction when using the Q5® Site-Directed Mutagenesis Kit?
11. How do I design primers to use with the Q5® Site-Directed Mutagenesis Kit?
12. What should I use for an annealing temperature with the Q5® Site-Directed Mutagenesis Kit?
13. I use the Q5 Site-Directed Mutagenesis Kit to introduce single mutations. How can I introduce multiple mutations?

Troubleshooting

• Be sure to use high-efficiency chemically-competent E. coli cells. The following competent E. coli cells have been shown to work with this kit:
  NEB #C2987, NEB 5-alpha (High Efficiency) (standard recommendation)
  NEB #C2992, NEB 5-alpha F´I^q (High Efficiency)
  NEB #C3019, NEB 10-beta (High Efficiency)
  NEB #C2984, NEB Turbo
  NEB #C2566, T7 Express
  NEB #C3029, Shuffle® T7
  
  For convenience, we offer a version of the Q5 Site Directed Mutagenesis Kit prepackaged with
  NEB #C2987 NEB 5-alpha Competent E. coli (High Efficiency) cells in product number NEB #E0554.
  
  Other chemically competent E. coli strains suitable for cloning can be substituted. Results will vary according to the quality and efficiency of the cells.
  
  
• Check that the transformation efficiency of the competent cells is ~1 x 10⁹ colony forming units (cfu) per μg. To calculate transformation efficiency, transform 2 μl of pUC19 DNA (NEB #N3041) (100 pg) into 50 μl of cells. Follow the transformation protocol on page 8. Prior to plating, dilute 10 μl of cells up to 1 ml in SOC. Plate 100 μl of this dilution. In this case, 150 colonies will yield a transformation efficiency of 1.5 x 10⁹ cfu/μg (μg DNA=0.0001, dilution=10/1000 x 100/1000).
• Ensure that your primers are designed properly. To take advantage of the exponential nature of the amplification reaction, the 5´ ends of the two primers should align back-to-back unless deletions are being made (see Figure 3). For best results, primers should be designed and annealing temperatures calculated using NEBaseChanger, the NEB online primer design software.
• Ensure there is a clean PCR product by visualizing 2–5 μl of the reaction on an agarose gel. Follow the suggestions below for low or impure PCR products.
• Only use 1 μl of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency. If the PCR yield is low, more product can be added to the KLD reaction, however a buffer exchange step, such as PCR purification, must be included prior to transformation.
• Only use 5 μl of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR purification, should be included prior to transformation.
• Ensure that the selectable marker in the plasmid matches the selection agent used in the plates
• Ensure the E. coli cells have been stored at –80°C.

• Ensure that the optimal annealing temperature (Ta) is used. High-Fidelity polymerases benefit from a Tm+3 annealing temp. Use NEBaseChanger, the NEB online primer design software, to calculate Ta. Alternatively, the optimal annealing temperature could be determined using a gradient PCR followed by agarose gel analysis.
• Ensure that the elongation time is adequate for the plasmid length. We recommend 20–30 seconds per kb of plasmid.
• Ensure that the final concentration of each primer is 0.5 μm. 
• Purify the primers with polyacrylamide gel electrophoresis (PAGE).

Resulting Plasmids Do Not Contain the Desired Mutation

• Ensure proper design of the mutagenic primers.
• Optimize the PCR conditions (see above).
• Use 1–25 ng of template in the PCR step. A small increase in the number of clones with no/incorrect mutation incorporated can occur if less than 1 ng or more than 25 ng of template is used.

Tech Tips

Citations & Technical Literature