Immunoprecipitation was performed by overnight incubation rotating in cold room and washes were performed. CTCF antibody (5 μl, Millipore 07-729) was combined with 50 μl of protein-A magnetic beads (Dynabeads) for one hour at room temperature, and added to sonicated chromatin from 15 million cells. Samples were sonicated on the bioruptor for 15 min (an agarose gel was performed to make sure that the sonicated DNA smear was in the range of 250–600 bp), supplemented with 1% Triton X-100 and centrifuged 5 min at 16,000 rcf at 4 ☌. Following ligation, nuclei were pelleted and resuspended in 350 μl cold Nuclei Lysis Buffer (50 mM Tris–HCl pH 7.5, 10 mM EDTA, 1% SDS, and 1x Protease Inhibitors) with incubation rotating in the cold room for 10 min. Ligation mix was added to the samples (150 μl 10x ligation buffer (NEB B0202S), 7.5 μl 20mg per ml BSA (NEB B9001S), 150 μl Triton X-100 10%, 4000 units T4 DNA ligase (NEB M0202S), and 655.5 μl H2O) for 4 h at RT with rotation. To fill in the restriction fragment overhangs and mark the DNA ends with biotin, 1.5 μl 10 mM dCTP, 1.5 μl 10 mM dGTP, 1.5 μl 10 mM dTTP, 37.5 μl 0.4 mM biotin-14-dATP (Life Technologies 19524-016), and 50 units Klenow (DNA polymerase I large fragment, NEB M0210L) were added to each tube, and incubated for 60 min at 37 ☌. MboI was inactivated by incubating the samples 20 min at 62 ☌. Chromatin was then digested by adding 50 μl of NEBuffer 2 10X and 350 units of MboI (NEB R0147M) at 37 ☌ for 2 h while rotating at 950 rpm. SDS was quenched by adding 285 μl of H2O and 50 μl of Triton X-100 10%, and incubating at 37 ☌ for 15 min. Cell pellets were collected, washed once in 500 μl ice-cold lysis buffer and then incubated in 100 μl 0.5% SDS at 62 ☌ for 10 min. Cells were then lysed in 500 μl ice-cold lysis buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630, protease inhibitor cocktail (Roche complete, EDTA-free)) rotating at 4 ☌ for 30 min. HiChIP was performed with 15 million cells, as per the original protocol54. Pellets were washed twice with ice-cold PBS, snap-frozen in liquid nitrogen and stored at −80 ☌. Cells were fixed in culture medium with 1% formaldehyde at RT for 10 min and quenched with 0.125 M glycine. The exo-Klenow fragment is used in some fluorescent labeling reactions for microarray, and also in dA and dT tailing, an important step in the process of ligating DNA adapters to DNA fragments, frequently used in preparing DNA libraries for Next-Gen sequencing.HiChIP was performed in triplicate (for NSD2 High: one KMS11 and two NTKO replicates from independent cultures, for NSD2 Low, two replicates from one clone and one replicate from the other one, from independent cultures). This form of the enzyme is called the exo- Klenow fragment. This results in forms of the enzyme being expressed that retain 5' → 3' polymerase activity, but lack any exonuclease activity (5' → 3' or 3' → 5'). This problem can be overcome by introducing mutations in the gene that encodes Klenow. Just as the 5' → 3' exonuclease activity of DNA polymerase I from E.coli can be undesirable, the 3' → 5' exonuclease activity of Klenow fragment can also be undesirable for certain applications. The Klenow fragment was also the original enzyme used for greatly amplifying segments of DNA in the polymerase chain reaction (PCR) process, before being replaced by thermostable enzymes such as Taq polymerase. Filling in receded 3' ends of DNA fragments to make 5' overhang blunt.Synthesis of double-stranded DNA from single-stranded templates.The Klenow fragment is extremely useful for research-based tasks such as: coli makes it unsuitable for many applications, the Klenow fragment, which lacks this activity, can be very useful in research. 5' → 3' polymerase activity, and 3' → 5' exonuclease activity).īecause the 5' → 3' exonuclease activity of DNA polymerase I from E. ![]() coli is cleaved by subtilisin retains the 5' → 3' exonuclease activity but does not have the other two activities exhibited by the Klenow fragment (i.e. The other smaller fragment formed when DNA polymerase I from E. First reported in 1970, it retains the 5' → 3' polymerase activity and the 3’ → 5’ exonuclease activity for removal of precoding nucleotides and proofreading, but loses its 5' → 3' exonuclease activity. coli is enzymatically cleaved by the protease subtilisin. The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. Functional domains in the Klenow Fragment (left) and DNA Polymerase I (PDB).
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