DNA Substrates
All our DNA substrates are available separately and have been prepared to a high standard for accurate quantitation of results. They are guaranteed for use with all our assay kits and enzymes.
Supercoiled pBR322 DNA
Supercoiled plasmid pBR322 DNA is produced by the large-scale alkaline-lysis method (Sambrook et al., 1989)1 from a high-copy number derivative of pBR322 (Boros et al., 1984)2.
It is further treated so that the DNA is more supercoiled and has a narrow range of linking numbers making it an ideal substrate for relaxation reactions.
It is shipped on dry ice at a concentration of 1mg/ml in TE (10mM Tris-HCl (pH 7.5), 1mM EDTA). Store at 4°C.
| Cat No. | Product | Description | Price |
|---|---|---|---|
| S5001 | Supercoiled pBR322 plasmid | 50 µg | £90 |
| S2502 | Supercoiled pBR322 plasmid | 250 µg | £330 |
| S5003 | Supercoiled pBR322 plasmid | 500 µg | £650 |
| S1004 | Supercoiled pBR322 plasmid | 1 mg | £1240 |
| POS5001 | Positively Supercoiled pBR322 plasmid | 50 µg | £160 |
| POS2502 | Positively Supercoiled pBR322 plasmid | 250 µg | £600 |
Relaxed pBR322 DNA
Supercoiled plasmid pBR322 DNA is produced by the large-scale alkaline-lysis method (Sambrook et al., 1989)1 from a high-copy number derivative of pBR322 (Boros et al.,1984)2 and is relaxed using chicken erythrocyte topoisomerase I (Trask and Muller, 1983)3.
It is shipped on dry ice at a concentration of 1mg/ml in TE (10 mM Tris-HCl (pH 7.5), 1 mM EDTA). Store at 4°C.
0.5 µg of relaxed pBR322 when incubated with 1 U of DNA gyrase in a reaction volume of 30 µl at 37°C for 30 minutes in incubation buffer is completely converted to the supercoiled form.
| Cat No. | Product | Description | Price |
|---|---|---|---|
| R5001 | Relaxed pBR322 plasmid | 50 µg | £90 |
| R2502 | Relaxed pBR322 plasmid | 250 µg | £330 |
| R5003 | Relaxed pBR322 plasmid | 500 µg | £650 |
| R1004 | Relaxed pBR322 plasmid | 1 mg | £1240 |
Linear pBR322 DNA
Supercoiled plasmid pBR322 DNA is produced by the large-scale alkaline-lysis method (Sambrook et al., 1989)1 from a high-copy number derivative of pBR322 (Boros et al., 1984)2.
It is linearised by incubation with EcoRI and further concentrated and purified to a final concentration of 1.0 mg/ml in TE (10mM Tris-HCl (pH 7.5), 1mM EDTA). Store at 4°C.
| Cat No. | Product | Description | Price |
|---|---|---|---|
| L0150 | Linear pBR322 | 150 µg | £70 |
| L0300 | Linear pBR322 | 300 µg | £130 |
| L0600 | Linear pBR322 | 600 µg | £255 |
| L1000 | Linear pBR322 | 1 mg | £405 |
kDNA (isolated from Crithidia fasciculata)
kDNA is purified from Crithidia fasciculata using the method described in Shapiro (Shapiro et al., 1999)4 and is supplied at a concentration of 100 ng/µl in 10 mM Tris-HCl (pH8.0), 1 mM EDTA. kDNA is an excellent substrate for use in topo IV assays and results can be obtained more quickly than those from relaxation assays because the decatenated mini circles can easily be resolved from the non-decatenated material which remains in the wells of the gel. Store at 4°C.
| Cat No. | Product | Description | Price |
|---|---|---|---|
| K2001 | kDNA | 20 µg | £85 |
| K1002 | kDNA | 100 µg | £330 |
| K2003 | kDNA | 200 µg | £490 |
| K4004 | kDNA | 400 µg | £740 |
References
- Sambrook, J., Fritsch, E.F. & Maniatis, T (1989). Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Press. Cold Spring Harbor, NY.
- Boros, I., Pósfai, G. & Venetianer, P. (1984). High-copy number derivatives of the plasmid cloning vector pBR322. Gene 30, 257-260
- Trask, D. K. & Muller, M. T. (1983) Biochemical characterization of topoisomerase I purified from avian erythrocytes. Nucleic Acids Res. 11, 2779-2800
- Shapiro, T.A., Klein, V.A. and Englund, P.T. (1999) Isolation of kinetoplast DNA, in DNA Topoisomerase Protocols Vol.I (Bjornsti, M-A and Osheroff, N., eds.). Humana Press Inc., N. Jersey, pp. 61-68