EZ Nucleosomal DNA Prep Kit
EZ Nucleosomal DNA Prep Kit
- For the isolation of nucleosome-associated DNA from mammalian and yeast cells.
- Ideal for use in nucleosome mapping studies.
The EZ Nucleosomal DNA Prep Kit is a streamlined procedure for the isolation of nucleosome-associated DNA. The kit includes reagents/procedures for the following: cell nuclei isolation, intact nuclei enzymatic digestion, and nucleosomal DNA purification. This kit includes two different enzymes for nucleosomal DNA preparation: Atlantis dsDNase and Micrococcal Nuclease. Enzymatic digestion yields very homogeneous populations of core nucleosomes and purification of the nucleosome-associated DNA is performed using Zymo Researchâ€™s proven spin column technology. The result is pure nucleosomal DNA ready for analysis in less than 45 minutes!
|Applicable For||All sensitive downstream applications such as qPCR and Next-Generation sequencing.|
|Processing Volume||≤106 cells|
|Purity||Typical A260/A280 ≥ 1.8|
|Sample Source||Nucleosome associated DNA isolation and purification from mammalian and yeast cells.|
|Sample Storage||Eluted DNA should be stored at ≤ -20°C.|
|Yield||Up to 25 µg total DNA can be eluted into ≥ 25 µl. For DNA 75 bp - 10 kb the recovery is 70-90%. For DNA 11 kb - 23 kb the recovery is 50-70%.|
Q1: What is the sensitivity of the enzyme to different cell types?
Once the nuclei are isolated, the enzyme sensitivity should be similar between different cell types. However, some cell lines will be more sensitive to the detergent in the Nuclei Prep Buffer and thus result in a loss of nuclei. You can dilute the Nuclei Prep Buffer 1:1 with dsDNase Digestion Buffer prior to nuclei isolation.
Q2: What is the difference between Atlantis dsDNase and MNase?
• Atlantis dsDNase is a double-stranded DNA specific endonuclease that cleaves phosphodiester bonds in DNA to yield oligonucleotides with 5’-phosphate and 3’-hydroxyl termini. Increasing the incubation time with Atlantis dsDNAse will increase efficiency and generate more mono-nucleosomal DNA.
• Micrococcal Nuclease (MNase) is a single and double-stranded DNA and RNA endonuclease. MNase is more specific for single-stranded nucleic acids, but cleavage is biased towards AT-rich and AU-rich sequences. MNase will result in more robust digestion compared to Atlantis dsDNase. Increasing the incubation time with MNase will increase efficiency of this enzyme and generate more mono-nucleosomal DNA.
• The enzyme selection depends on desired downstream applications.
Q3: Do the sticky-end fragments have 5’ vs. 3’-overhangs? What is the relative abundance of different ends?
Atlantis dsDNAse generates random-end, i.e. both blunt- and sticky-end DNA fragments and there is no specific length for overhangs.
Q4: What if the nucleosomal DNA is not shearing properly and the majority of DNA is still intact?
Washing the trypsinized cell pellets with PBS is very critical step. Residual EDTA in the cell pellets will decrease both Atlantis dsDNAse and MNase digestion efficiency. Other considerations for incomplete shearing: • Check that the correct number of cells was digested. (D5220 kit: 1x106 mammalian cells) • If you only want mono-nucleosomal DNA instead of nucleosomal ladder containing mono-, di-, tri-nucleosomal, etc., you will need to increase the enzyme concentration. Based on our experience, we need to use >1U per Atlantis dsDNAse per million cells.
Micrococcal nuclease digestion was performed on embryonic stem cells using the EZ Nucleosomal DNA Prep Kit and paired-end libraries were generated for sequencing. Research showed heterogeneous chromatin organization around transcription start sites as well as unique nucleosome profiles associated with epigenetic patterns that can identify pluripotency.Yazdi PG, et. al. (2015). Nucleosome Organization in Human Embryonic Stem Cells. PLoS ONE. 10(8).
The EZ Nucleosomal DNA Prep Kit was used by researchers to isolate nucleosomal DNA from 293FT cells prior to performing Chromatin Accessibility Real Time PCR (CHART-PCR) to investigate the IL-2 promoter chromatin architecture in the presence of TATA-box binding protein (TBP-TALE) and VP64-TALE activators in non-immune cells. They found that the combination of AD’CF TALEs but not empty vector control increased DNase I hypersensitivity across the IL-2 promoter.Anthony K et al. (2014) Activation of Silenced Cytokine Gene Promoters by the Synergistic Effect of TBP-TALE and VP64-TALE Activators. PLoS One. 2014 Apr 22;9(4):e95790.
Researchers used the EZ Nucleosomal DNA Prep Kit from Zymo Research to isolate nuclei, digest chromatin, and purify nucleosomal DNA from Hep3B cells or SW13 cells that were transfected with either an empty vector or vectors expressing WT BRG1 or ATPase-dead BRG1. Purified nucleosomal DNA was used for SYBR Green–based qPCR to determine nucleosome positioning on the CA9 and LDHA promoters.Sena JA et al. (2013) BRG1 and BRM chromatin remodeling complexes regulate the hypoxia response by acting as a co-activator for a subset of HIF target genes. Mol Cell Biol.
|D5220-3||MN Digestion Buffer||50 ml||€36,00|
|D5220-4||5X MN Stop Buffer||6 ml||€14,00|
|D5220-2||Nuclei Prep Buffer||50 ml||€36,00|
|D5220-1||Micrococcal Nuclease||10 U/100 µl||€27,50|
|E2030-1||Atlantis Digestion Buffer||50 ml||€37,00|
|D4003-2-6||DNA Wash Buffer (Concentrate)||6 ml||€10,00|
|D4003-1-25||DNA Binding Buffer||25 ml||€21,00|
|D3004-4-1||DNA Elution Buffer||1 ml||€12,00|