Mutation Generation System™ Kit (MGS™ Kit)

Transposon-mediated system for insertion scanning mutagenesis:
A powerful new approach for functional analyses of proteins and regulatory DNA regions.

Description

The Mutation Generation System™ Kit (MGS™ Kit) is designed for rapid construction of insertion mutation libraries for any kind of DNA clones. The system employs the highly efficient transposition machinery of the bacteriophage Mu to generate a pool of 15 bp insertion mutants that can be utilized in a variety of functional analyses of the encoded proteins or regulatory DNA regions.

Applications

The MGS Kit generates random fifteen basepair in vitro insertions into any target DNA for:

• Rapid generation of in-frame five amino acid insertion libraries of any protein for functional analyses
  
• Rapid and random mutagenesis of cloned promoters and other regulatory DNA regions
  
• Random insertion of a NotI restriction enzyme site into any target DNA clone

Transposition reaction into linear target DNA

Advantages

• Thousands of different insertion clones from a single reaction
  
• Generates random insertions of 5 amino acids in all 3 reading frames
  
• Short in-frame insertions; no stop codons
  
• Flexibility in mapping mutants of interest: mutations are easily mapped by NotI or PCR*.
• Faster and more effective than linker scanning mutagenesis

Components

The kit contains sufficient materials for 10 reactions.

Kit components
MuA Transposase	10 µl	(0.22 µg/µl in MuA storage buffer)
Entranceposon (M1-CamR)	10 µl	(100 ng/µl in TE buffer)
Entranceposon (M1-KanR)	10 µl	(100 ng/µl in TE buffer)
5x Reaction Buffer for MuA Transposase	100 µl
Control Target DNA	10 µl	(370 ng/µl in TE buffer)
NotI Miniprimer	50 µl	(25 µM in dd water)

Storage stability

Store the components at –20 °C. Stable for 1 year from the date of packaging when stored and handled properly.

System protocol

Day 1. Perform the transposition reaction, in which MuA Transposase catalyzes the in vitro transposition reaction by inserting the Entranceposons into the target DNA plasmids at random sites (70 min). Transform the DNA into competent E.coli cells, plate and cultivate the cells overnight.

Day 2. Pool colonies from the transformation plates for plasmid DNA preparation.

Digest the plasmid DNA with the restriction enzyme NotI to remove the body of the Entranceposon. Self-ligate the NotI-digested plasmids. This will result in a 15 bp insertion in the target DNA plasmid. Transform, plate and cultivate the cells overnight.

Day 3. Pool colonies from the transformation plates for plasmid DNA preparation. Alternatively: map the Entranceposon insertions in individual clones using colony-PCR and make plasmid DNA preparations from the clones of your choice.

The 15 bp insertion in a target gene is translated into five extra amino acids in the encoded protein.

Ordering information

Mutation Generation System™ Kit (MGS™ Kit )
F-701	Mutation Generation System™ Kit (MGS™ Kit)	For 10 reactions
F-750	MuA Transposase with 5x Reaction Buffer	20 µl (0.22 µg/µl in MuA storage buffer)
F-760	Entranceposon (M1-CamR)	10 µl (100 ng/µl in TE buffer)
F-762	Entranceposon (M1-KanR)	10 µl (100 ng/µl in TE buffer)