Point mutations, deletion/insertion of a fragment
  • OVERVIEW

Point mutations, deletion/insertion of a fragment

Seattle Genova offers a variety of mutagenesis services such as introducing point mutations, insertions, or deletions into your gene of interest. You can either just let us know your gene sequences or send us your plasmid to mutate. When the work is done, we can clone the verified sequence into your choice of any vector backbones to meet your needs of cloning, expression, virus production and many other applications.

Point-mutations can be introduced to plasmids using primers (with the desired mutation) in a PCR protocol that amplifies the entire plasmid template. The parent template is removed using a methylation-dependent endonuclease (i.e. DpnI), and bacteria are transformed with the nuclease-resistant nicked plasmid (the PCR product). Plasmids are isolated from the resulting colonies, and screened for the desired modification. Finally, the positive clones are sequenced to confirm the desired modification and the absence of additional modifications.


The major steps of a point mutation are:

· Primer Design

Design a 11 bp of complementary sequence on either side of the desired mutation.

· PCR

Amplify the entire plasmid template with PCR using high fidelity enzyme.

· Screening

If a restriction site was introduced (or ablated), bacterial colonies can be screened by identifying the presence (or absence) of that particular site with fragment length polymorphism (RFLP) analysis.

· Validating

Final validation is performed by sequencing functional regions of the plasmid including the insert.


Deliverables

· 10 μg of mutated plasmid

· Project report including primer design and mutation methods

· Sequencing data

Point mutations, deletion/insertion of a fragment

Seattle Genova offers a variety of mutagenesis services such as introducing point mutations, insertions, or deletions into your gene of interest. You can either just let us know your gene sequences or send us your plasmid to mutate. When the work is done, we can clone the verified sequence into your choice of any vector backbones to meet your needs of cloning, expression, virus production and many other applications.

Point-mutations can be introduced to plasmids using primers (with the desired mutation) in a PCR protocol that amplifies the entire plasmid template. The parent template is removed using a methylation-dependent endonuclease (i.e. DpnI), and bacteria are transformed with the nuclease-resistant nicked plasmid (the PCR product). Plasmids are isolated from the resulting colonies, and screened for the desired modification. Finally, the positive clones are sequenced to confirm the desired modification and the absence of additional modifications.


The major steps of a point mutation are:

· Primer Design

Design a 11 bp of complementary sequence on either side of the desired mutation.

· PCR

Amplify the entire plasmid template with PCR using high fidelity enzyme.

· Screening

If a restriction site was introduced (or ablated), bacterial colonies can be screened by identifying the presence (or absence) of that particular site with fragment length polymorphism (RFLP) analysis.

· Validating

Final validation is performed by sequencing functional regions of the plasmid including the insert.


Deliverables

· 10 μg of mutated plasmid

· Project report including primer design and mutation methods

· Sequencing data

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