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Stop Generation System™ Kit (STOP™ Kit)
The transposon based STOP™ Kit for generating truncated proteins for functional assays: saturated libraries of mutated proteins in a single reaction
Description
Transposon-based STOP™ Kit is developed for functional analysis of proteins. This new transposon tools enable the creation of saturated libraries of mutated proteins in a single reaction. The STOP Kit Entranceposons contain translational stop codons in all three reading frames within the terminal portion of the transposon sequence. The proprietary modification of the Stop Generation System™ Kit makes it possible to generate a saturated C-terminal deletion library from virtually any target protein with a maximum addition of three amino acids. The transposon-based method requires less hands-on time than any other method, thousands of mutated clones are ready for expression studies in just two days.
Applications
The STOP Kit generates truncated proteins for functional assays of:
- Enzymes
- Receptors
- Structural proteins etc.
Transposition reaction into linear target DNA
Advantages
- Saturated library of truncated proteins from a single reaction in two days
- Translational STOP codon in all three reading frames
- The target DNA sequence can be unknown
- Faster and more effective than conventional methods
- No specific primers required
Components
The kit contains sufficient materials for 10 reactions.
Kit components |
|||
|---|---|---|---|
MuA Transposase |
10 µl |
(0.22 µg/µl in MuA storage buffer) |
|
Entranceposon (STOP-KanR) |
10 µl |
(100 ng/µl in TE buffer) |
|
5x Reaction Buffer for MuA Transposase |
100 µl |
|
|
DMSO 100 % |
500 µl |
|
|
Control Target DNA |
10 µl |
(370 ng/µl in TE buffer) |
|
MuEnd-2 Primer |
50 µl |
(25 µM in dd water) |
|
SeqE Primer |
250 ul |
(10 uM in dd water) |
|
SeqW Primer |
250 ul |
(10 uM 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 with the transposition reaction mixture. Plate out on selection plates (LB+kanamycin) and cultivate the cells overnight.
Time required: appr. 3 h
Hands-on time: max. 1 h
Figure 1. In vitro transposition reaction components.
Figure 2. In vitro transposition reaction. (Incubate 60 min at 30 °C, Heat inactivate 10 min at 75 °C)
Day 2. Pool the colonies from transformation plates and prepare plasmid DNA from the pool. Transform the expression host cells with the plasmid pool.
Time required: appr. 3 h
Hands-on time: appr. 2 h
Result: library of truncated proteins ready for expression studies.
Ordering information
| Stop Generation System™ Kit (STOP™ Kit) | |
|---|---|
F-703 |
For 10 reactions |
