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Phusion® Site-Directed Mutagenesis Kit > FAQ |  | Phusion® Site-Directed Mutagenesis Kit FAQ
See the DNA Modifying Enzymes and Cloning FAQ also.
Q1: How does the Phusion® Site-Directed Mutagenesis Kit destroy the wild-type template?
Q2: What plasmid sizes can be amplified using the Phusion® Site-Directed Mutagenesis Kit?
Q3: Do I need to purify my plasmid before or after ligation when using the Phusion® Site-Directed Mutagenesis Kit?
Q4: What strain should I use for the transformation step when using the Phusion® Site-Directed Mutagenesis Kit?
Q5: Are there particular requirements for designing primers to use with the Phusion® Site-Directed Mutagenesis Kit?
Q6: What should I use for an annealing temperature with the Phusion® Site-Directed Mutagenesis Kit?
Q7: What is the most common reason for incorrect clones when screening mutants?
Q8: Why do I not see any PCR product when using the Phusion® Site-Directed Mutagenesis Kit?
Q9: Why is my PCR product missing nucleotides at the ligation site?
Q10: Why is the desired mutation missing from the transformants that I screened?
Q1: How does the Phusion® Site-Directed Mutagenesis Kit destroy the wild-type template?
A1: There is no need to destroy the background because it is low due to the exponential amplification, unlike linear amplifications.
Q2: What plasmid sizes can be amplified using the Phusion® Site-Directed Mutagenesis Kit?
A2: Up to 10 kb plasmids can be amplified successfully.
Q3: Do I need to purify my plasmid before or after ligation when using the Phusion® Site-Directed Mutagenesis Kit?
A3: No, purification is not necessary, and Quick T4 DNA Ligase is included for fast ligation.
Q4: What strain should I use for the transformation step when using the Phusion® Site-Directed Mutagenesis Kit?
A4: Phusion SDM is compatible with all strains of competent E.coli cells.
Q5: Are there particular requirements for designing primers to use with the Phusion® Site-Directed Mutagenesis Kit?
A5: The primer design depends on the type of the desired mutation. Both primers do not have to be mutagenic. However, if two separate mutations are desired, two mutagenic primers are needed. The primers must be phosphorylated at the 5' end. We recommended using commercially phosphorylated primers. However, a protocol for primer phosphorylation is included in the kit manual. It is crucial that only full-length (n) molecules of the primers are present in the reaction mixture. The presence of shorter primers (n-1, n-2 etc.), which lack nucleotides at the 5'; end, will lead to shorter PCR products and thus to missing nucleotides at the ligation site. Therefore, it is recommended only to use primers purified with reverse phase high performance liquid chromatography (RP-HPLC) or with polyacrylamide gel electrophoresis (PAGE). For primers longer than 40 nucleotides, purification with PAGE is preferable.
Point mutations
Point mutations are created by designing a mismatch in the mutagenic primer.There can be more than one mismatch in the mutagenic primer, either separated by correctly matched nucleotides or present in consecutive nucleotides. For generating point mutations, the length of the correctly matched sequence in the mutagenic primers should be on average 24-30 nucleotides. The desired mutation should be in the middle of the primer with 10-15 perfectly matched nucleotides on each side. Primers should be designed so that the 5' ends of the forward and reverse primers are abutting.
Deletions
Deletions are created by designing primers that border the deleted area on both sides. To generate a deletion, the primers should be perfectly matched on their entire length, which should be 24-30 nucleotides.
Insertions
For generating insertions, primers can be designed in two alternative ways.
1. For longer insertions, a stretch of mismatched nucleotides is designed in the 5'end(s) of one or both primers. If mismatched stretches are designed in the 5' ends of both primers, they form one entire insertion when the ends of the PCR product are ligated. The Tm’s should be calculated for the perfectly matched portion of the primers. Insertions of 50 bp (25 bp on each primer) or less are recommended when using this method.
2. For short insertions, a stretch of mismatched nucleotides is designed in the middle of the primer. The length of the correctly matched sequence in the mutagenic primers should be on average 24-30 nucleotides. The desired insertion should be in the middle of the primer with 10-15 perfectly matched nucleotides on each side. In both cases, primers should be designed so that the 5' ends of the forward and reverse primers are abutting.
Q6: What should I use for an annealing temperature with the Phusion® Site-Directed Mutagenesis Kit?
A6: Phusion® Hot Start II DNA Polymerase has the ability to stabilize primer-template hybridization. Note that the optimal annealing temperature for Phusion Hot Start II DNA Polymerase may differ significantly from that of Taq-based polymerases. Always use the Tm calculator and instructions on Finnzymes’ website to determine the Tm values of primers and optimal annealing temperature. For primers longer than 20 nucleotides, use an annealing temperature 3°C higher than the Tm of the lower Tm primer given by the calculator. If the primer length is 20 nucleotides or less, use an annealing temperature equal to the Tm of the lower Tm primer given by the calculator. It is recommended to design primers so that the annealing temperature falls between 65°C and 72°C. In case the annealing temperature approaches 72°C, a two-step cycling protocol without a separate annealing step can be used when running the PCR. Note that the optimal annealing temperature may differ from the instructions above, if there are mismatches in the middle of the primer sequence. Some experimental optimization may be required.
Q7: What is the most common reason for incorrect clones when screening mutants?
A7: The most common reason for incorrect clones is incomplete primers, which result in lacking nucleotides a the ligation site. However, these clones generally do contain the desired mutation.
Q8: Why do I not see any PCR product when using the Phusion® Site-Directed Mutagenesis Kit?
A8: *Pipetting errors are common due to the high glycerol content of the storage buffer.
*Plasmid template concentration may be too low.
*Extension time may be too short.
*Annealing temperature may be too high.
*Primers phosphorylated with T4 polynucleotide kinase must be purified after phosphorylation.
*Correct primer design is crucial.
Q9: Why is my PCR product missing nucleotides at the ligation site?
A9: When using the Phusion® Site-Directed Mutagenesis Kit, the primers must be complete and purified after phosphorylation.
Q10: Why is the desired mutation missing from the transformants that I screened?
A10: An excessive amount of target plasmid in the PCR can result in background transformants. Also, some DNA structures, including inverted and tandem repeats, are selected against by E.coli. Some recombinant proteins are not well tolerated by E.coli and can result in selection pressure against mutation.
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