dsDNA Shearase Plus is an endonuclease that cleaves phosphodiester bonds in DNA to yield oligonucleotides with 5’-phosphate and 3’-hydroxyl termini. It has a particularly strong preference for dsDNA and generates random-ended DNA fragments of the desired size in a single step. This enzyme is compatible with low volume inputs, thus minimizing sample loss.
Q1: Is there any sequence preference at the cutting sites?
Yes, we observe that there is more “T” in the second base. Only the first and second base are a little imbalanced, but the nucleotide distribution is balanced from the 3rd base onward.
Q2: Can reaction be scaled up/down?
The genomic DNA: enzyme ratio is the most critical step for dsDNA Shearase Plus reaction. It is necessary to scale up or down proportionally the amount of enzyme when adjusting the DNA input.
- 5X Reaction Buffer
- Template DNA
- Volume
- 250 ng (x µl)
- 500 ng (x µl)
- 1 µg (x µl)
- Water
- Volume
- 7 µl – x µl
- 14 µl – x µl
- 28 µl – x µl
- dsDNA Shearase Plus
- Total Volume
1 U is defined as the amount of enzyme required to convert 250 ng human DNA to 100-500 bp in 20 min (1 U is 2 µl).
Q3: How can I ensure the enzyme has been inactivated?
The enzyme can be inactivated by incubation at 65°C for 5 minutes. The DNA Clean & Concentrator-5 (D4003) can be used to clean up the reaction.
Q4: Can AMPure beads be used to clean up the reaction?
Yes, please follow the manufacturer’s protocol. You can also use the Select-a-Size DNA Clean & Concentrator MagBead Kit.
Q5: Can RNA be used as a substrate for dsDNA Shearase Plus?
dsDNA Shearase Plus is dsDNA specific, thus, RNA will remain intact if there is RNA contamination in the samples. Hybridization between DNA and RNA might occur in RNA contaminated samples, reducing the performance and efficiency of the fragmentation. We recommend using RNA-free DNA as a substrate.
Q6: Is the enzymatic digestion affected by DNA methylation?
Enzymatic activity is not affected by methylation. Enzymes have been tested with human, mouse, and plant DNA and the performance was the same.
Q7: Why is the enzyme not shearing properly?
Here are some possible reasons:
• The dsDNA Shearase Plus is specific for dsDNA and does not work efficiently for ssDNA.
• The performance of the enzyme is significantly affected by RNA contamination present in the samples Check the quality of your DNA sample as DNA/RNA hybridization might occur during the 42°C incubation.
• Make sure that enzyme is scaled up or down properly in the reaction.
