| Recognition site | GTT↑AAC
CAA↓TTG |
| Source | An E.coli strain, that carries the cloned gene Hpa I from Haemophilus parainfluenzae |
| Assayed on | Lambda DNA, plasmid DNA |
| Description | Turbo Hpa I is used for short time (15 min) DNA digestion in SE-Buffer Y .
Applications:
- Fast DNA analysis
- Fast preparation of vectors for cloning
- Double digestion
|
| Reaction buffer | SE-buffer Y (33 mM Tris-acetate (pH 7.9 at 25°C); 10 mM magnesium acetate; 66 mM potassium acetate; 1 mM DTT.) |
| Optimal temperature | 37oC |
| Storage conditions | 10 mM Tris-HCl (pH 7.5); 50 mM NaCl; 0.1 mM EDTA; 1 mM DTT; 200 μg/ml BSA; 50% glycerol. Store at -20°C. |
| Ligation | After digestion with 1 μl of Turbo Hpa I, approximately 60% 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 Y.
|
| Methylation sensitivity | not tested |
| Inactivation 20 minutes under | 65oC |
| Notes | Enzyme Properties:
1 μl of Turbo Hpa I cuts 1 μg of DNA in 1x SE-Buffer Y in 15 min (assayed on Lambda 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 Y - 2 μl
DNA - 0.2-1 μg
Nuclease-free water - to 20 μl
+ 1 μl of Turbo Hpa I
Mix by pipette tip carefully.
Incubate at 37°C for 15 min.
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| References: | Sharp, P.A., Sugden, B., Sambrook, J. Biochemistry 12: 3055-3063 (1973).
Garfin, D.E., Goodman, H.M. Biochem. Biophys. Res. Commun. 59: 108-116 (1974).
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