Site specific mutagenesis is; a technique used to:
- create specific alterations in a gene
- study the relationship between protein structure and its function
- modify or repair DNA constructs
- alter sites of restriction, insert or delete elements of a vector (for example tag elements, or exchange of promoters)
- We can introduce any type of single or multiple mutation * (substitution, insertion or deletion) in plasmids up to 15kb.
THE SERVICE INCLUDES:
- oligo-sense sequencing of the portion to be mutagenized to confirm DNA before mutagenesis
- design and synthesis of the mutagenic oligo
- DNA amplification
- transformation, cell culture and purification of 5 random colonies
- sequencing of purified clones to confirm mutation
- 4/5 working days for single mutation
- sending of alignment with the wt control sequence and single electropherogram of the positive clone
- dispatch of 1/4 µ g of purified mutated plasmid DNA
- sequencing of the entire insert or plasmid with synthesis of the relative oligos that will be stored in the personal Oligo Bank
- control of the plasmid by digestion with restriction enzymes
- major preparation of purified plasmid DNA (midi or maxi)
- sequencing of the site to be mutagenized and of the positive clone also with anti-sense oligo.
It should be noted that the completion of all quality controls provided, while not guaranteeing the functioning of the clone, will in any case give Bio-Fab the right to receive the agreed payment up to the stage of the project carried out.
MATERIAL TO BE SHIPPED TO THE LABORATORY:
- 2 µg of purified plasmid containing the DNA to be mutated, from a strain of E. coli dam +. Plasmids isolated from dam- strains are not suitable (e.g. jm110, scs110 and BL21)
- plasmid map with indication of antibiotic resistance and target DNA sequence
- mutagenesis instructions: list and localization of the bases for insertion, deletion and replacement
- form completed in its entirety
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