Feb 14, 2022
Version 2
Q5® Site-Directed Mutagenesis (E0554) V.2
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
External link: https://www.neb.com/protocols/2013/01/26/q5-site-directed-mutagenesis-kit-protocol-e0554
Protocol Citation: New England Biolabs 2022. Q5® Site-Directed Mutagenesis (E0554). protocols.io https://dx.doi.org/10.17504/protocols.io.bddfi23nVersion created by New England Biolabs
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 07, 2020
Last Modified: February 14, 2022
Protocol Integer ID: 33927
Keywords: SDM, Q5, site directed mutagenesis, exponential amplification for Q5, PCR
Abstract
This is the protocol for the Q5® Site-Directed Mutagenesis Kit (E0554).
Guidelines
DESCRIPTION
The Q5® Site-Directed Mutagenesis Kit enables rapid, site-specific mutagenesis of double-stranded plasmid DNA in less than 2 hours (Figure 1). The kit utilizes the robust Q5 Hot Start High-Fidelity DNA Polymerase along with custom mutagenic primers to create insertions, deletions and substitutions in a wide variety of plasmids. After PCR, the amplified material is added directly to a unique Kinase-Ligase-DpnI (KLD) enzyme mix for rapid (5 minutes), room temperature circularization and template removal (Figure 2). Transformation into high-efficiency NEB 5-alpha Competent E. coli, provided with the kit, ensures robust results with plasmids up to at least 20 kb in length.
Figure 1: Site-specific mutagenesis proceeds in less than 2 hours.
The use of a master mix, a unique multi-enzyme KLD enzyme mix, and a fast polymerase ensures that, for most plasmids, the mutagenesis reaction is complete in less than two hours.
Figure 2: Q5 Site-Directed Mutagenesis Kit Overview.
This kit is designed for rapid and efficient incorporation of insertions, deletions and substitutions into doublestranded plasmid DNA. The first step is an exponential amplification using standard primers and a master mix fomulation of Q5 Hot Start High-Fidelity DNA Polymerase. The second step involves incubation with a unique enzyme mix containing a kinase, a ligase and DpnI. Together, these enzymes allow for rapid circularization of the PCR product and removal of the template DNA. The last step is a high-efficiency transformation into chemicallycompetent cells (provided).
Figure 3: Primer Design for the Q5 Site-Directed Mutagenesis Kit
Substitutions, deletions and insertions are incorporated into plasmid DNA through the use of specifically designed forward (black) and reverse (red) primers. Unlike kits that rely on linear amplification, primers designed for the Q5 Site-Directed Mutagenesis Kit should not overlap to ensure that the benefits of exponential amplification are realized. A) Substitutions are created by incorporating the desired nucleotide change(s) (denoted by *) in the center of the forward primer, including at least 10 complementary nucleotides on the 3´side of the mutation(s). The reverse primer is designed so that the 5´ ends of the two primers anneal back-to- back. B) Deletions are engineered by designing standard, non-mutagenic forward and reverse primers that flank the region to be deleted. C) Insertions less than or equal to 6 nucleotides are incorporated into the 5´ end of the forward primer while the reverse primer anneals back-to-back with the 5´ end of the complementary region of the forward primer. D) Larger insertions can be created by incorporating half of the desired insertion into the 5´ ends of both primers. The maximum size of the insertion is largely dictated by oligonucleotide synthesis limitations.
TROUBLESHOOTING
No/Low Colonies
- 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 NEB 5-alpha Competent E. coli cells have been stored at -80° C.
- 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 the provided control pUC19 DNA (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).
No/Low PCR Product
- 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 10–20 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.
NOTES
Storage Note:
The Q5 Site-Directed Mutagenesis Kit is stable at –80°C for one year. For convenience, the Q5 Hot Start High-Fidelity 2X Master Mix, KLD Enzyme Mix, KLD Reaction Buffer, Control Primers and Template DNA are packaged together in a separate box that can be removed and stored at –20°C for two years with no loss of activity. The SOC can be removed and stored at room temperature. It is important to store the NEB 5-alpha Competent E. coli at –80°C, and avoid repeated freeze-thaw cycles.
REFERENCES
1. Kalnins et al., (1983). The EMBO Journal. 2, 593-597.
2. Dickinson DJ, Ward JD, Reiner DJ, Goldstein B. (2013). Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination.. Nat Methods. Sep 1, PubMedID: 23995389
Materials
MATERIALS
Q5 Site-Directed Mutagenesis Kit - 10 rxnsNew England BiolabsCatalog #E0554S
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Exponential Amplification (PCR)
Exponential Amplification (PCR)
Assemble the following reagents in a thin-walled PCR tube.
| A | B | C | |
| 25 μl RXN | FINAL CONC. | ||
| Q5 Hot Start High-Fidelity 2X Master Mix | 12.5 μl | 1X | |
| 10 μM Forward Primer | 1.25 μl | 0.5 μM | |
| 10 μM Reverse Primer | 1.25 μl | 0.5 μM | |
| Template DNA (1–25 ng/μl) | 1 μl | 1-25 ng | |
| Nuclease-free water | 9.0 μl |
Mix reagents completely.
Transfer to a thermocycler and perform the following cycling conditions:
Thermocycling Conditions for a Routine PCR:
| A | B | C | |
| STEP | TEMP | TIME | |
| Initial Denaturation | 98°C | 30 seconds | |
| 25 Cycles | 98°C | 10 seconds | |
| 50–72°C* | 10–30 seconds | ||
| 72°C | 20–30 seconds/kb | ||
| Final Extension | 72°C | 2 minutes | |
| Hold | 4–10°C |
* For a Q5-optimized annealing temperature of mutagenic primers, please use NEBaseChanger™, the online NEB primer design software. For pre-designed, back-to-back primer sets, a Ta = Tm + 3 rule can be applied, but optimization may be necessary.
Kinase, Ligase & DpnI (KLD) Treatment
Kinase, Ligase & DpnI (KLD) Treatment
Assemble the following reagents:
| A | B | C | |
| VOLUME | FINAL CONC. | ||
| PCR Product | 1 μl | ||
| 2X KLD Reaction Buffer | 5 μl | 1X | |
| 10X KLD Enzyme Mix | 1 μl | 1X | |
| Nuclease-free Water | 3 μl |
Mix well by pipetting up and down.
Incubate at Room temperature for 00:05:00 .
Transformation
Transformation
Thaw a tube of NEB 5-alpha Competent E. coli cells On ice .
Add 5 µL KLD mix from the "KLD Section" above to the tube of thawed cells.
Carefully flick the tube 4-5 times to mix. Do not vortex.
Place the mixture On ice for 00:30:00 .
Heat shock at 42 °C for 00:00:30 .
Place On ice for 00:05:00 .
Pipette 950 µL room temperature SOC into the mixture.
Incubate at 37 °C for 01:00:00 with shaking (250 rpm ).
Mix the cells thoroughly by flicking the tube and inverting.
Spread 50 µL -100 µL onto a selection plate.
Incubate Overnight at 37 °C .
Note
It may be necessary (particularly for simple substitution and deletion experiments) to make a 10- to 40-fold dilution of the transformation mix in SOC prior to plating, to avoid a lawn of colonies.