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
250 ng (x ?l)
500 ng (x ?l)
1 ?g (x ?l)
7 ?l ? x ?l
14 ?l ? x ?l
28 ?l ? x ?l
dsDNA Shearase Plus
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 ?l).
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.
MULTIPLEXED ELIMINATION OF WILD-TYPE DNA AND HIGH RESOLUTION MELTING PRI?
Genomic DNA shearing was performed with the use of dsDNA Shearase Plus (Zymo Research) in a total 10?l reaction (1x dsDNA Shearase Plus Reaction Buffer, 100ng of biopsies and matched blood samples were collected from patients with metastatic breast cancer consented to Dana-Farber Cancer Institute IRB protocol #05-246. . Genomic DNA shearing was performed with the use of dsDNA Shearase Plus (Zymo Research) in a total 10?l reaction (1x dsDNA Shearase Plus Reaction Buffer, 100ng of genomic DNA and 1 unit of dsDNA Shearase Plus enzyme).. Genomic DNA was quantified with Qubit 3.0 fluorometer (Life Technologies).
Epigenetic activation of meiotic recombination near Arabidopsis thaliana?
DNA shearing was carried out for 20 min at 37?C with 0.4 units of DNA Shearase (Zymo Research).DNA was extracted from three rosette leaves of 5-wk-old plants and 150 ng of DNA used as input for each library.. DNA shearing was carried out for 20 min at 37?C with 0.4 units of DNA Shearase (Zymo Research).. Each set of 96 libraries was sequenced on an Illumina NextSeq 500 (300-cycle Mid Output run).
MBD2 upregulates miR-301a-5p to induce kidney cell apoptosis during vanc?
Briefly, purified genomic DNA was sheared to fragments of 200 bp using DNA Shearase (Zymo Research), which were used for methylated CpG immunoprecipitation. CpG-DNA immunoprecipitation assay was carried out according to the manufacturer?s instructions (Zymo Research, Orange County, CA, USA) as previously described. . Briefly, purified genomic DNA was sheared to fragments of 200 bp using DNA Shearase (Zymo Research), which were used for methylated CpG immunoprecipitation.. Methylated DNA was recovered and subjected to PCR analysis on an ABI Onestepplus Real-Time PCR system (Shanghai, China).
?I believe our whole lab wants to switch to your shearase to put an end to all the sonicating we are currently doing for ChIP, MeDIP, and MeDIP-seq experiments.?
– Garrett K (The University of Alabama at Birmingham)
- Catalog#: E2018-50 / E2018-200
- Package Length (in Inches): 1.8 /0
- Package Width (in Inches): 0.4 /0
- Package Height (in Inches): 0.4 /0
- Package Weight (in Pounds): 0.1 /0
- Size: 50 U /200 U
- Unit Standard: Metric
- Volume Units: Milliliters