New England Biolabs, E0554S, Q5® Site-Directed Mutagenesis Kit

The Q5 Related Categories sgRNA Synthesis,, DNA Assembly, Cloning and Mutagenesis Kits Applications Site Directed Mutagenesis,, Site Directed Mutagenesis FAQ Q: How do I design primers to use with the Q5® Site-Directed Mutagenesis Kit? A: For best results, back-to-back primers should be designed using our online primer design software, which can be found at: NEBaseChanger.neb.com. For general primer design guidelines, follow the instructions below. Note that both primers do not have to be mutagenic and do not have to be phosphorylated or purified. Substitutions Substitutions are created by designing a mismatch in the center of the mutagenic primer. Include at least 10 nts that are complementary to your plasmid at the 3' end of the primer. To accommodate large mutations (from 7 to 50 per primer), changes should be incorporated at the 5’end of the mutagenic primer. The 5' end of the second primer will begin at the base next to the 5' end of the first primer and proceed in the opposite direction on the complementary strand. This primer can be 100% complementary to the plasmid sequence or can contain mismatches, if desired. The absence of any overlap ensures that exponential (rather than linear) amplification will take place. Deletions Deletions are created by designing primers that flank both sides of the area to be deleted. The two primers should be designed in opposite directions with their 5’ ends adjacent to the area to be deleted. The primers can be 100% complementary to the plasmid sequence or can contain mismatches and/or insertions if desired. Insertions The sequence to be inserted should be added to the 5' end of the mutagenic primer. For insertions >6 nts, the insertion sequence can be split between the two primers. Half of the insert should be added to the 5' end of the forward primer and the other half should be added to the 5' end of the reverse primer. As described for substitutions, there should be at least 10 nts that are complementary to your plasmid on the 3' end of each primer. The maximum size of the insertion is largely dictated by oligo synthesis limitations. Insertions of up to 100 nts (50 nts at the 5’ end of each primer) can routinely be accommodated using this kit. Note: For primers greater than 60 nts long, it is recommended to have them purified by either HPLC or PAGE. Q: What should I use for an annealing temperature with the Q5® Site-Directed Mutagenesis Kit? A: We recommend using the NEB online primer design tool, NEBaseChanger, to provide an optimized annealing temperature for mutagenic primer pairs. For predesigned back-to-back primer sets, a Tm+3 rule can be applied but optimization may be necessary. Note that mismatches in the mutagenic primer are not accounted for using standard Tm calculations and the optimal annealing temperature for Q5 High Fidelity Hot Start DNA Polymerase may differ significantly from that of Taq-based polymerases. For primer pairs with annealing temperatures close to 72°C, a two-step cycling protocol without a separate annealing step can be used. Q: Do I need to purify my plasmid before or after the KLD reaction when using the Q5® Site-Directed Mutagenesis Kit? A: No, purification is not necessary. Q: What plasmid sizes can be amplified using the Q5® Site-Directed Mutagenesis Kit? A: We have not yet found the upper limit for plasmid size. To date, plasmids up to 20 kb have been amplified successfully. Q: What is the maximum number of nucleotides that can be inserted with this kit? A: Due to the back-to-back primer design, insertions up to 100 nucleotides can routinely be tolerated by appending up to 50 nucleotides to the 5’ end of each primer. Note that HPLC or PAGE purification is recommended for any primer greater than 60 nts. Q: What is the maximum distance that can be tolerated between substitutions? A: Due to the back-to-back primer design, mutations within 100 nucleotides of each other can routinely be made in one step. Note that HPLC or PAGE purification is recommended for any primer greater than 60 nts. Q: Typically, what percentage of transformants will have the desired mutation incorporated? A: Due to the high-fidelity polymerase and robust KLD treatment step, the frequency of desired mutations is quite high (>90% for most experiments). When undesired mutations are observed, they are typically at the plasmid ligation site. Q: What is the KLD Mix? A: The KLD Mix contains a blend of kinase, ligase and DpnI enzymes in a buffer that preserves the activity of the enzymes. This formulation allows efficient phosphorylation, intramolecular ligation/circularization and template removal in a single 5 minute reaction step at room temperature. Q: Can I use my own competent cells? A: Yes. Although the Q5® Site-Directed Mutagenesis Kit will work optimally with the supplied high-efficiency NEB competent E. coli, other chemically competent E. coli strains suitable for cloning can be substituted. Results will vary according to the quality and efficiency of the cells. Q: If I double my PCR size, should I add more PCR mix to the KLD reaction? A: No. The 10X KLD Enzyme Mix is formulated to work well with 1 μl of the PCR mix regardless of the volume of the PCR. Q: Why is the desired mutation missing from the transformants that I screened? A: Although this is not a common result, there are a few known causes for background transformants: 1) excessive plasmid in the PCR, 2) selection against specific DNA structures such as inverted or tandem repeats, and 3) selection against the recombinant protein by the host E. coli cells. Q: Why do I not see my PCR product after using the Q5® Site-Directed Mutagenesis Kit? A: Correct primer design and annealing temperature determination are very important. Use of the NEB online site-directed mutagenesis primer design tool, NEBaseChanger, for design and annealing temperature determination is highly recommended. In addition, ensure that 1) the plasmid template concentration is between 1 and 25 ng, 2) the final primer concentration is 0.5 µM, and 3) the extension time is 20-30 seconds per kb of plasmid. Q: I use the Q5 Site-Directed Mutagenesis Kit to introduce single mutations. How can I introduce multiple mutations? A: NEB® has developed a protocol using NEBuilder HiFi DNA Assembly Master Mix to simplify the construction of multiple site-directed mutagenesis. The technique involves the design of complimentary flanking primers to align fragments. This application note describes the use of the NEBuilder HiFi DNA Assembly Master Mix to generate multiple site-directed mutagenesis at the same time. We recommend 18-20nt overlaps but the length of the primers can vary. The primers are completely overlapping as illustrated below. The goal is to achieve balanced Ta’s for the primer pairs. The mutations are positioned in the middle of the primer. There must be enough bases on either side, and most importantly 3’ of the mutation, so that the primer will anneal properly to the template. Q: There is a precipitate in the bottom of the Master Mix tube? Is this normal? A: Q5® Master Mix may precipitate upon freezing and thawing. This does not indicate a problem with the product. For optimal performance, thaw fully and resuspend any precipitate by warming to room temperature and inverting gently until completely dissolved. Q: Can I perform a gel extraction if my PCR product has multiple bands? A: Yes. If PCR results in off-target amplification, it's best to optimize the reaction as much as possible. If PCR cannot be optimized, we recommend performing a gel extraction using the Monarch® Spin DNA Gel Extraction Kit (NEB #T1120). Up to 4 µl of purified DNA can be carried into a KLD reaction. For help optimizing PCR, see these guidelines: https://www.neb.com/en-us/tools-and-resources/usage-guidelines/guidelines-for-pcr-optimization-with-thermophilic-dna-polymerases