For the RCA reaction, the solution including ligated product was added to 2–50 pmol of biotin-primer, 0.2 mM dNTP mix, 20 U NxGen®phi29 DNA polymerase, 0.05 U pyrophosphatase, and 0.8 mM DTT in phi29 reaction buffer to a final volume of 50 μl and incubated at 37 °C for 30 min. The RCA product was characterized by polyacrylamide gel electrophoresis. The solution including RCA product was purified using Illustra Microspin G-25 columns (Cytiva), to remove the enzyme, DTT, and buffer components. The purified product was then mixed with 1–100 pmol of FAM-probe to a final volume of 10 μl and heated to 95 ℃ for 10 min, followed by cooling to 25 ℃ and incubation for 20 min for hybridization of the FAM-probe to the amplified target DNA produced by RCA.
Illustra microspin g 25 columns
Manufactured by Cytiva
The Illustra Microspin G-25 columns are size-exclusion chromatography columns designed for the rapid and efficient purification of biomolecules. The columns are pre-packed with Sephadex G-25 resin, which allows for the separation of small molecules from larger biomolecules such as proteins, peptides, and nucleic acids.
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Lab products found in correlation
2 protocols using illustra microspin g 25 columns
1
Amplification and Detection of DNA Targets
2
Protein Purification and Crystallization
Check if the same lab product or an alternative is used in the 5 most similar protocols
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For crystallization trials,
protein purification tags were removed by overnight cleavage with
TEV protease at 30 °C as previously described.40 (link) Cleaved proteins were purified by affinity-tag purification
using a HisTrap FF crude column (Cytiva) on an ÄktäPure
FPLC instrument, collecting the flow-through. Proteins were further
separated by size exclusion chromatography (HiLoad 26/600 Superdex
75, Cytiva) and concentrated using Ultra-4 or 15 mL centrifugal filter
devices (Amicon, Merck). The correct size and high purity were verified
via SDS–PAGE and LC-MS analysis. Protein labeling was performed
in activity buffer, overnight at room temperature using fluorophore
substrates at 10 μM (CA-TMR/CA-CPY and BG-TMR for HT7/HOB and
SNAP, respectively) in the presence of 5 μM (3 mg) protein.
After concentration to ∼200 μL, an excess of fluorophore
substrate was removed by buffer exchange using Illustra microspin
G-25 columns (Cytiva) according to the manufacturer’s instructions.
Protein labeling was verified by SDS–PAGE fluorescence scanning
and LC-MS analysis. Protein concentrations were adjusted between 10
and 20 mg/mL and submitted to crystallization trials using different
commercial screens via mixing in a 200 nL final volume protein solution/crystallization
solution (1:1) using a Mosquito robot (TTP Labtech).
protein purification tags were removed by overnight cleavage with
TEV protease at 30 °C as previously described.40 (link) Cleaved proteins were purified by affinity-tag purification
using a HisTrap FF crude column (Cytiva) on an ÄktäPure
FPLC instrument, collecting the flow-through. Proteins were further
separated by size exclusion chromatography (HiLoad 26/600 Superdex
75, Cytiva) and concentrated using Ultra-4 or 15 mL centrifugal filter
devices (Amicon, Merck). The correct size and high purity were verified
via SDS–PAGE and LC-MS analysis. Protein labeling was performed
in activity buffer, overnight at room temperature using fluorophore
substrates at 10 μM (CA-TMR/CA-CPY and BG-TMR for HT7/HOB and
SNAP, respectively) in the presence of 5 μM (3 mg) protein.
After concentration to ∼200 μL, an excess of fluorophore
substrate was removed by buffer exchange using Illustra microspin
G-25 columns (Cytiva) according to the manufacturer’s instructions.
Protein labeling was verified by SDS–PAGE fluorescence scanning
and LC-MS analysis. Protein concentrations were adjusted between 10
and 20 mg/mL and submitted to crystallization trials using different
commercial screens via mixing in a 200 nL final volume protein solution/crystallization
solution (1:1) using a Mosquito robot (TTP Labtech).
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