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| | | | TnsABC* Transposase | | | Discontinued as of 4/1/09. Product is no longer available. | |
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Description:
TnsABC* Transposase is used in the GPS™ series of systems (NEB #E7100S, #E7101S, #E7102S) to insert the Transprimer element from pGPS plasmids randomly into any desired target DNA in vitro (1-3).
Source:
The three component proteins are purified separately from E. coli K-12 strains containing plasmids encoding TnsA, TnsB and TnsC* (2).
Applications:- Transposition of the Tn7-based transposons used in the GPS Systems
Reagents Supplied:
GPS™ Buffer Pack (10X)
Start Solution
Reaction & Storage Conditions
Reaction Conditions:
1X GPS™ Buffer
Supplemented with 15 mM Start Solution
Incubate at
37°C.
1X GPS™ Buffer:
25 mM Tris-HCl
2 mM ATP
2 mM Dithiothreitol
pH 8.0 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme necessary to result in a minimum of 1% of target molecules gaining a Transprimer insertion in a standard GPS control reaction using 0.08 μg of the 2.8 kb control plasmid.
Concentration:
1,000 units/ml
Storage Conditions:
25 mM Tris-HCl
500 mM NaCl
2 mM MgCl2
1 mM ATP
0.5 mM Dithiothreitol
0.8 mM EDTA
50% Glycerol
pH 7.9 @ 25°C
Storage Temperature:
-20°C
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
GPS Control Reaction::
A 20 µl GPS Control Reaction using 0.02 µg pGPS1.1, 0.08 µg Control Target plasmid and 1 µl TnsABC* Transposase in 1 X GPS Buffer supplemented with 15 mM Start Solution after 10 minutes then incubated for 1 hour at 37°C and transformed into chemically-compentent E. coli (1 x 107 per µg pBR322), resulted in > 1000 colonies being obtained after spreading on Kan plates and incubating overnight at 37°C. > 1% of target molecules acquired a Transprimer.
16-Hour Incubation::
A 50 µl reaction containing 1 µg of Hind III fragments of λ DNA and 4 µl of TnsABC* Transposase in 1X GPS Buffer supplemented with 15 mM Start Solution resulted in no alteration to the sharp characteristic banding pattern of the DNA substrate after a 16 hour incubation at 37°C.
Exonuclease Activity::
Incubation of 2 µl of enzyme with 1 µg sonicated 3H DNA (105 cpm/µg) for 4 hours at 37°C in 50 µl reaction buffer supplemented with 15 mM Start Solution released 0% radioactivity.
Endonuclease Activity::
Incubation of 4 µl of enzyme with 1 µg of ΦX174 RF I DNA for 4 hours at 37°C in 50 µl 1X GPS Buffer supplemented with 15 mM Start Solution resulted in < 5% conversion to RF II.
References
- Craig, N.L. (1996) Curr. Top. Microbiol. Immunol., 204, 27-48.
- Stellwagen, A.E. and Craig, N.L. (1997) Genetics, 145, 573-585.
- Biery, M.C., Stewart, F.J., Stellwagen, A.E., Raleigh, E.A. and Craig, N.L. (2000) Nucl. Acids Res., 28, 1067-1077.
Legal
Patents:
Johns Hopkins University: Licensed Under U.S. Patent No. 6,420,524
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