|
|
|
 |
|
Prices are in US dollars and valid only for US orders.
|
Description:
USER™ (Uracil-Specific Excision Reagent) Enzyme (1) is supplied with the USER Friendly Cloning Kit (NEB #E5500). The enzyme generates a single nucleotide gap at the location of a uracil. USER Enzyme is a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII. UDG catalyses the excision of a uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact (2,3). The lyase activity of Endonuclease VIII breaks the phosphodiester backbone at the 3´ and 5´ sides of the abasic site so that base-free deoxyribose is released (4,5).
Source:
The two component proteins are purified separately from E. coli K-12 strains containing plasmids encoding Endonuclease VIII and Uracil-DNA Glycosylase.
Enzyme Properties
Heat Inactivation:
No
Reaction & Storage Conditions
Incubate at
37°C.
Unit Definition:
One unit is defined as the amount of enzyme required to nick 10 pmol of a 34 mer oligonucleotide duplex containing a single uracil base in a total reaction volume of 10 μl in 15 minutes at 37°C in 1X T4 DNA Ligase Buffer containing 10 pmol of flourescently labeled oligonucleotide duplex.
Concentration:
1,000 units/ml
Storage Conditions:
12.5 mM Tris-HCl
33 mM KCl
33 mM NaCl
1 mM Dithiothreitol
0.3 mM EDTA
160 µg/ml BSA
50% Glycerol
pH 7.7 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- USER Enzyme is active in all commercial PCR buffers tested. It also has 100% activity in TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA). For further information, see the USER Friendly Cloning Kit manual (NEB #E5500).
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Transformation Reaction:
A standard USER Reaction was performed as described in Appendix V of the USER Friendly Cloning Kit (NEB #E5500) manual [20 ng linearized pNEB206A, 1 µl USER Enzyme and 10 µl (100 ng) of a 950 bp control PCR product amplified using Taq DNA Polymerase and primers containing uracil, designed as recommended in the USER Friendly Cloning Kit manual]. After transformation into chemically-competent cells (NEB ER2267 at 5 x 106 c.f.u./µg pNEB206A), 50 µl of the 1 ml outgrowth was spread on Amp + Xgal + IPTG plates. Greater than 95% of colonies were white (i.e., contained recombinant molecules).
References
- Bitinaite, J., unpublished observations.
- Lindhal, T., Ljungquist, S., Siegert, W., Nyberg, B. and Sperens, B. (1977) J. Biol. Chem., 252, 3286-3294.
- Lindhal, T. (1982) Annu. Rev. Biochem., 51, 61-64.
- Melamede, R.J., Hatahet, Z., Kow, Y.W., Ide, H. and Wallace, S.S. (1994) Biochemistry, 33, 1255-1264.
- Jiang, D., Hatahet, Z., Melamede, R.J., Kow, Y.W. and Wallace, S.S. (1997) J. Biol. Chem., 272, 32230-32239.
Companion Products
Universal USER Cassette
USER Friendly Cloning Kit
|
