- What is the difference between dsDNase and HL-dsDNase?
- What is the advantage of ArcticZymes heat labile DNases over non heat labile DNases from other manufacturers?
- What might be the disadvantage of the heat lability of a DNase?
- Can I replace DNase I with (HL-)dsDNase in all applications?
- Does (HL-)ds DNase cleave single-stranded DNA?
- Does (HL-)dsDNase digest DNA of DNA-RNA hybrid?
- Can (HL-)dsDNase be used for removal of DNA from RNA preps?
- Can I use (HL-)dsDNase for PCR carry over prevention?
- What is the reaction temperature of (HL-)dsDNase?
- What salt concentration is optimal for (HL-)dsDNase?
- What pH is optimal for (HL-)dsDNase?
- What is the recommended (HL-)dsDNase amount per reaction?
- What is the molecular weight of (HL-)dsDNase?
- What is the minimal DNA length required for HL-ds DNase cleavage?
- How long is the products created by the HL-dsDNase after cleavage?
- Is (HL-)dsDNase a tagged protein?
- Is (HL-)dsDNase free from E. coli and human DNA?
- Does the (HL-)dsDNase preparation contain BSA?
- How stable is the (HL-)dsDNase?
- What are the recommendations for storage of (HL-)dsDNase?
- How many freeze -thaw cycles can (HL-)dsDNase tolerate?
- Dry ice was melted / I left my vial at room temp, can I still use the dsDNase?
HL-dsDNase is a modified version of dsDNase for improving heat-lability. In standard PCR buffers, dsDNase is inactivated by 15 min incubation at 65˚C while HL-dsDNase is inactivated after 5 min at 58˚C above pH 8.0.
Because of lower inactivation temperature, HL-dsDNase is recommended when RNA is present during inactivation.
What is the advantage of ArcticZymes heat labile DNases over non heat labile DNases from other manufacturers?
A heat-labile DNase does not need to be physically or chemically removed from the reaction before downstream processing, as Heat-inactivation completely and irreversibly removes its activity.
Our heat-labile dsDNases are not suited when activity above 50˚C (dsDNase) or 45˚C (HL-dsDNase) is necessary.
Our dsDNases are not suited when the assay has to be performed above 45°C.
(HL-)dsDNase is double strand DNA specific, whereas DNaseI has activity against ssDNA also (1-5 % compared to dsDNA).If the application is dependent of the small activity against ssDNA, (HL-)dsDNase will not be suitable.
(HL-)dsDNase is heat-labile. If removing DNA at temperatures above 50-60˚C is needed, (HL-)dsDNase will not be suitable.
No – It cleaves only doble-stranded DNA. However, if ssDNA forms double-stranded sections internally or with other ssDNA present during treatment, it will act as a target for (HL-)dsDNase. Because of this, longer and complex ssDNA (for instance cDNA) will in most cases be partially degraded by (HL-)dsDNase.
(HL-)dsDNase may under the right conditions digest the DNA from DNA:RNA hybrids. However, the activity towards the DNA-strand in a DNA-RNA hybrid is about 20 %of the activity towards dsDNA. The RNA-strand in a hybrid is not cleaved.
In RT-PCR reactions, HL-dsDNase is not active during reverse transcription if the RT incubation step is at 50˚C or above. However, reverse transcriptases do not have hot-start capabilities, and cDNA synthesized at temperatures lower than 40˚C will be a target for HL-dsDNase (confer question5 above for details).
Yes – HL-dsDNase is most suited for this purpose because of its lower inactivation temperature. Take care to consider the amount of Mg in the cleavage mix to avoid autocatalysis of RNA.
For removal of gDNA from RNA preps we recommend the Heat&Run gDNA removal kit.
Yes – (HL-)dsDNase can be used to remove any contaminating dsDNA from PCR mastermixes , including carry-over contamination. (HL-)dsDNase is not relying on the use of dUTPs in PCR for being effective in carry-over prevention, making it possible to use standard nucleotides in combination with contamination control. It is not possible to do carry-over prevention in any solution containing any double-stranded template, as (HL-)dsDNase would digest also the template.
We recommend the PCR decontamination kit for this purpose.
(HL-)dsDNase is active in the temperature range 20-50˚C, with optimum activity at 40˚C.
(HL-)dsDNase needs above 2.5mM Mg2+ for activity. The activity increases with higher Mg2+ concentration up to 10 mM Mg2+. As EDTA chelates free Mg2+, (HL-)dsDNase is inhibited by EDTA.
(HL-)dsDNase is less active in buffers containing >10mM KCl/NaCl. Concentrations above 50 mM should be avoided.
(HL-)dsDNase is inhibited by (NH4)2SO4.
The optimum activity for (HL-)dsDNase is at pH 7.5.
In general we recommend to use 2U (HL-)dsDNase per 50µl reaction. If high pH, salt or other inhibiting factors are present, increased amounts of (HL-)dsDNase might be beneficial in order to compensate for lost activity.
The molecular weight of the enzymes is 42 kD.
The minimal substrate, or minimal length of DNA to be a substrate for (HL-)dsDNases is about 9-10 bp.
The end-products generated by the enzymes are distributed around 5 bp ± 3 bp.
No, (HL-)dsDNase is not a tagged protein.
Yes, as far as we have tested, we have no indication that the enzymes contain any amplifiable DNA.
No, the (HL-)dsDNase contains no BSA.
(HL-)dsDNase is stable for years if stored at recommended conditions, at -20˚C.
(HL-)dsDNase is relatively stable if stored at 4˚C. However, this is normally not recommended for more than 6 months.
(HL-)dsDNase can withstand 5-7 days incubation at room temp (21-25°C) without significant loss of activity.
(HL-)dsDNase is very unstable at temperatures above 50˚C.
(HL-)dsDNase is stable at -20˚C for at least two years and at least 6 months at 4˚C.
We have seen no significant effect on enzyme activity upon a 5x freeze/thaw cycle.
(HL-)dsDNase can withstand 5-7 days incubation at room temp (21-25°C) without significant loss of activity. However, stability decreases as the temperature increases. Thus, prolonged incubations at temperatures above 25°C could negatively affect the activity.