|Bsp19 I|| |
|Cat. #||E047T||E048T |
|Number of reactions||50||250 |
|Volume, μl||50||250 |
|Source||Bacillus species 19|
|Assayed on||Lambda DNA, plasmid DNA|
Turbo Bsp19 I is used for a short time (10 min) of 1 μg DNA digestion in SE-Buffer W+ in 50 μl of reaction mixture.
- Fast DNA analysis
- Fast preparation of vectors for cloning
- Double digestion
|Reaction buffer||SE-buffer 2W+ (20 mM Tris-HCl (pH 8,5 at 25°C); 10 mM MgCl2; 200 mM NaCl; 1 mM DTT ; 100 μg/ml BSA)|
|Storage conditions||10 mM Tris-HCl (pH 7.5); 50 mM KCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 200 μg/ml BSA; 50% glycerol. Store at -20°C.|
|Ligation||After digestion with 1 μl of Turbo Bsp19 I, approximately 90% of the DNA fragments can be ligated with high-activity T4 DNA Ligase and recut.|
|Non-specific hydrolisis||No detectable degradation of a single-stranded and double-stranded oligonucleotide was observed after incubation with 1 μl of restriction endonuclease for 3 hours.
|Reagents Supplied with Enzyme||
10 X SE-buffer 2W+
|Methylation sensitivity||Bsp19I cuts hemimethylated site |
and doesn't cut methylated
|Inactivation 20 minutes under||65oC|
1 μl of Turbo Bsp19 I cuts 1 μg of DNA in 1x SE-Buffer W+ in 10 min (assayed on Lambda DNA and plasmid DNA). A short time of DNA digestion requires high quality purification of DNA sample (PCR fragments should be purified after amplification).
Please note that supercoiled plasmid DNA and PCR fragments may have varying rates of cleavage and sometimes need more time to be completely digested.
Standard protocol of Turbo reaction:
20 μl of the reaction volume:
10x SE-Buffer W+ - 2 μl
DNA - 0.2-1 μg
Nuclease-free water - to 20 μl
+ 1 μl of Turbo Bsp19 I
Mix by pipette tip carefully.
Incubate at 37°C for 10 min.
|References:||Repin, V.E., Rechkunova, N.I., Degtyarev, S.Kh. Unpublished observations.
Repin, V.E., Lebedev, L.R., Andreeva, I.S., Puchkova, L.I., Zernov, Y.P., Serov, G.D., Tereshchenko, T.A., Aphinogenova, G.N., Pustoshilova, N.M.
Biotekhnologiya 0: 18-27 (1998).
Chernukhin V.A, Abdurashitov M.A., Tomilov V.N., Gonchar D.A., Degtyarev S.Kh. Comparative analysis of mouse chromosomal DNA digestion with restriction endonucleases in vitro and in silico // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.3, No 4, pp 19-27, 2007