The NEBaseChanger® Tool makes designing back-to-back primers for using the Q5® Site-Directed Mutagenesis Kit a quick and easy process. To begin select the new sequence button, then paste your starting or wild type plasma sequence in raw or FASTA format. Alternatively, you can choose from one of the common vector sequences available in the dropdown menu. If desired, you can add a sequence name to the file, which will populate on the summary. Next, set the mutagenesis type by choosing substitution, insertion, or deletion from the options on the right. For substitutions and deletions, select the nucleotides to be changed or deleted. For insertions, select the nucleotides that flank the desired insertion site. You may click and drag within the displayed sequence to select a range.
You may also use the find sequence field to search within your plasma for your region of interest. Once this is found, you can select by clicking and dragging over the highlighted area. When making a selection, the first and last values will then populate in the start and end fields at the right. For substitutions and insertions, enter the new or inserted sequence into the desired sequence box beneath the start and end positions. For deletions, this is not needed.
If inserting a tag, you can select from the common peptide tags listed in the dropdown menu beneath the desired sequence box. NEBBaseChanger will generate primer sequences, recommended annealing temperature, and display the three frame amino acid sequence corresponding to the mutation. Once the primers are designed, we would suggest checking them against your full starting plasma sequence for any alternative binding sites. This can help ensure a single specific PCR product following the amplification step.
The annealing temperature suggested by NEBaseChanger takes into account the specific conditions of NEB's Q5 High Fidelity DNA Polymerase. It is not uncommon for the primer Tms shown in NEBaseChanger to be higher than what is seen from an oligo manufacturer upon receipt. It is recommended to use the specific Tas suggested by NEBaseChanger. You can use the Get Summary feature to generate a document that contains the desired primer information along with the sequence of the final construct and recommended protocol. NEBaseChanger is available at nebasechanger.neb.com. To learn more about the Q5 SDM Kit, please view this video.
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