About ChIP DNA Clean & Concentrator™
The Chromatin Immunoprecipitation (ChIP) DNA Clean & Concentrator™ provides a hassle-free method for the rapid purification and concentration of high quality DNA from any step in a standard ChIP protocol. This includes samples that have undergone reverse cross-linking, Proteinase K or RNase A digestion, mechanical or nuclease-mediated DNA shearing, and samples eluted from chromatin-antibody-bead complexes. The specially formulated ChIP DNA Binding Buffer promotes DNA adsorption to the column in the presence of detergents, antibodies, and proteinases that are often used for ChIP. This kit may be applied to any routine ChIP procedure to determine DNA concentration of samples that have undergone reverse cross-linking following DNA shearing. It can also be used for the removal of TES, 0.1M NaHCO3 and 1% SDS from DNA eluted from chromatin-antibody-bead complexes and can be used to purify DNA from buffers containing up to 1% SDS or 5% NP-40, Tween-20, Triton X-100 or Sarkosyl.
|DNA Recovery||For DNA 50 bp to 10 kb the recovery is 70-90%. For DNA 11 kb to 23 kb the recovery is 50-70%.|
|DNA Size Limits||50 bp to ~ 23 kb|
|Sample Sources||Wherever DNA isolation and purification is required during standard ChIP protocols. This includes samples that have undergone reverse cross-linking and Proteinase K or RNase A digestion following either 1) mechanical or nuclease-mediated DNA shearing or 2) elution from chromatin-antibody-bead complexes in TES, 0.1M NaHCO3 and 1% SDS, or other buffers containing up to 1% SDS. This kit can also be used for DNA purification from PCR, enzymatic digestion, kinase, phosphatase and other enzymatic reactions.|
|DNA Purity||High quality, purified DNA is eluted with elution buffer or water and is especially well suited for PCR amplification, arrays, Southern blot analysis, DNA quantification, sequencing, and other molecular applications.|
|Product Detergent Tolerance||≤ 5% Triton X-100, ≤ 5% Tween-20, ≤ 5% Sarkosyl, ≤ 1% SDS|
Agarose gel electrophoresis of DNA isolated from cell lysates. High quality DNA can be efficiently recovered from Saccharomyces cerevisiae cell lysates using the ChIP DNA Clean & Concentrator™. Duplicate purifications were performed with 5, 15, and 30 µl cell lysate and an equal volume of eluted DNA was loaded into each lane. The size marker M1 and M2 are 100 bp and 1 kb ladders, respectively (Zymo Research).
In a 1.5 ml microcentrifuge tube, add 5 volumes of ChIP DNA Binding Buffer to each volume of sample (5:1). Mix briefly.
Example 1: Add 250 µl ChIP DNA Binding Buffer to 50 µl cell lysate following DNA shearing, reverse cross-linking and Proteinase K digestion in TES (50 mM Tris-Cl, pH 8.0, 10 mM EDTA, 1% SDS) or 0.1M NaHCO3 containing 1% SDS .
Example 2: Add 600 µl ChIP DNA Binding Buffer to 120 µl eluent in TES or 0.1M NaHCO3 containing 1% SDS buffers from chromatin-antibody-Protein A agarose-bead complexes followed by reverse cross-linking and Proteinase K digestion.
Transfer mixture to a provided Zymo-Spin™ Column in a Collection Tube.
Centrifuge at ≥ 10,000 x g for 30 seconds. Discard the flow-through.
Add 200 µl Wash Buffer to the column. Centrifuge at ≥ 10,000 x g for 30 seconds. Repeat wash step.
Add 6-100 µl Elution Buffer directly to the column matrix. Transfer the column to a new 1.5 ml microcentrifuge tube and centrifuge at ≥ 10,000 x g for 30 seconds to elute the DNA.
The ChIP DNA Clean & Concentrator™ was used to purify the DNA fragments in DNA–protein complexes precipitated by antibodies against KLF6 or Sp1. Purified DNA was used to analyze the capacity of KLF6 and Sp1 for binding to exon 1 of the tmsg-1 gene.
ChIP assays were carried out from Xenopus embryonic tissues and DNA fragments were purified using ChIP DNA Clean & Concentrator. Purified DNA was used to study the occupancy of TCF proteins on Vent2 promoter.
ChIP DNA was recovered and purified using Zymo's ChIP DNA Clean and Concentrator™ Kit. Purified ChIP DNA was subjected to TaqMan qPCR with specific primers and probes to study the effect of 5-aza-CdR treatment on the association of AP-2alpha at hZip1 and hZip3 promoter regions.
Proteinase K/RNase-treated DNA fragments were purified using ChIP DNA Clean & Concentrator™ spin column and purified DNA was used to analyze the association of VDR and changes of histone H3 and H4 acetylation within the major regulatory modules of the CYP3A4 gene.