Genomic DNA (gDNA) was sonicated into 200- to 400-bp-long fragments (Covaris). Briefly, 5-hmC labeling reactions were performed in a 20 µL solution containing 50mM HEPES buffer (pH 7.9), 25 mM MgCl2, 100 ng/µL sonicated gDNA (200–400 bp), 250 µM UDP-6-N3-Glu, and 2.25 µM βGT enzyme. The reactions were incubated for 1 h at 37°C, and following incubation, the labeled DNA was purified by the QIAquick PCR Purification Kit (Qiagen) and eluted in water. The click chemistry reaction was performed by the addition of 150 µM dibenzocyclooctyne modified biotin into the eluted DNA, and the reaction mixture was incubated for 2 h at 37°C. The DNA samples were then purified using the MinElute Reaction Cleanup Kit (Qiagen), and the amount of eluted DNA was determined by Nanodrop UV spectroscope (Thermo). hMe-Seal (affinity pulldown of 5-hmC) was performed as described previously (Song et al. 2011 (link)). In total, 20 μg of gDNA was labeled in 30 µL with a biotin linker that contained a disulfide bond. Labeled DNA was pulled down with streptavidin-coated magnetic beads (Invitrogen). After washing, captured DNA was released from beads with 50 mM DTT; excess DTT was removed by chromatography spin column (Bio-Rad), and the DNA was purified in a total volume of 12 μL by the MinElute Reaction Cleanup Kit (Qiagen). The final yield of pulled-down DNA was determined using PicoGreen (Invitrogen).
Chromatography spin column
Manufactured by Bio-Rad
The Chromatography spin column is a laboratory equipment used for the separation and purification of biomolecules, such as proteins, nucleic acids, and other macromolecules. It functions by utilizing centrifugal force to pass a sample through a porous matrix, allowing for the selective retention and elution of the desired components.
Automatically generated - may contain errors
Lab products found in correlation
Picogreen, by Thermo Fisher Scientific (1 mentions)
Streptavidin coated magnetic beads, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Minelute reaction cleanup kit, by Qiagen (1 mentions)
Hek293t cells, by Mirus Bio (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Transit lt1 transfection reagent, by Mirus Bio (1 mentions)
Anti flag affinity gel, by Merck Group (1 mentions)
3 protocols using chromatography spin column
1
Genome-wide profiling of 5-hmC
2
Transient Antibody Expression in HEK 293T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For transient transfections, HEK 293T cells (ATCC) were grown in Dulbecco’s modification of Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 100 U of penicillin and 100 mg of streptomycin per ml. DMEM supplemented with 10% FBS. To express recombinant antibodies, 7.5 ug of each matched pairs of heavy and light chain expression vectors cloned from a single cell were co-transfected into HEK 293T cells in 10cm dishes using Mirus Transit-LT-1 transfection reagent (Mirus). The following day, supernatants were removed and replaced with 10 ml of EX-CELL 293 serum free media (SAFC Biosciences). Supernatants containing expressed antibodies were harvested 5–6 days later and cleared of cell debris by centrifugation at 1,000g for 10 minutes. Antibodies were purified by addition of 25ul of Protein G agarose beads (Pierce) and incubated with rotation overnight at 4°C. The beads were then pelleted by centrifugation at 800g for 5 minutes, resuspended in 200 ul PBS and transferred to a chromatography spin column (Bio-Rad) equilibrated with PBS. The beads were washed 2 times with PBS, then eluted in 3 fractions of 200 ul each with 0.1M glycine (pH3.0) into tubes containing 20 ul of 1M Tris (pH8.0). Sodium azide was then added to a final concentration of 0.1%.
3
Affinity Purification of FLAG-Tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
50 mM HEPES-KOH (HEPES, Sigma-Aldrich, #H3375-1 KG KOH, Sigma Millipore, # 1050121000)
150 mM NaCl, Sigma-Aldrich, # 746398-5 KG
5 mM EDTA: Sigma-Aldrich, # 607-429-00-8
1% Triton X-100: Sigma, #X100-1L
FLAG Peptide Elution Buffer: 50 mM HEPES, 500 mM NaCl, pH 7.4
Protease Inhibitor Tablets; Complete, Mini, EDTA-free Protease Inhibitor Cocktail, Sigma-Aldrich cat. no. 5892953001
Anti-FLAG affinity gel; Sigma-Aldrich, cat. no. A2220-5 ML
3X FLAG peptide; Sigma-Aldrich cat. no. F4799-4 MG
Chromatography spin column; Bio-Rad cat. no. 7326204
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!