Mutant Primers

Primer design for semi-random mutagenesis

Description

This tool designs site-directed mutagenesis primer pairs in bulk based on specified amino acid mutations required. This includes options for degerate codons (see below).

The primer pairs can be made to be fully overlapping like traditional QuikChange primers or a partially overlapping primer pair strategy (Xia et al., 2015 ).

As the traditional Quikchange protocol (Agilent) works poorly for codon mutants, a better option is to have the primers with an user-defined overlap length ( e.g. 22 bp) centred around the codon to mutate and will have a 3’ overhand long enough to allow the region beyond the mutagenized codon to anneal with the template above a given melting temperature, while taking into account terminal GC clamp.

If the overlap is in the 20–30 bases range, the primers can be also be used for Gibson assembly.

Input

To make primers at the ends of the coding sequence, the neighbouring noncoding sequence are required. Two options are available:

To give the upstream and downstream sequence separately.
To give a sequence with a start and end position. A variant of this is to give the starting/end position as a short sequence to match as opposed to a number.

Upstream
Sequence
Sequence
Downstream
Sequence
First base
Last base

Add to the following textarea the list of mutations desired, such as A13P. If more complex mutations, such as degenerate codons are to be use mark the destination codon in square brakets after the AA residue and its number, e.g. A13[NNK]. For mutations to multiple amino acids (e.g. A13GP) please specify the required codon for now. If considering degenerate codons and are not sure which to pick, try using the helper tool.



Mutation
List
Enzyme
Target Tm
°C
GC clamp bonus
°C
Method
Overlap
nt
Length