DNA replication kit

$1,199

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The DNA replication kit is suitable to kick start your single-molecule experiments to study the kinetics of enzyme activities involved in DNA replication. The biotinylated pKYB1 DNA containing  a 25-nt 5′-overhang serves to load DNA polymerase enzymes (e.g., T7 DNA polymerase) to investigate the polymerization dynamics, proofreading activity, and pause states of DNA replication in real time.

Overall, the DNA replication kit will provide you with a ready-to-go system for single-molecule applications to:

  1. Obtain kinetics and mechanistic data of the different phases of the DNA replication process at the single-molecule level with millisecond and base-pair resolution.
  2. Correlate different tensions applied to a DNA – mimicking nucleotide miss-incorporation – with polymerization rates, exonucleolytic activity, and pause states of DNA polymerase enzymes.
  3. Test polymerase activity with the u-Flux™ Laminar Flow Microfluidics that, through its multiple channels, facilitates switching between different experimental conditions.

A schematic illustration depicting the exonucleolytic activity of T7 DNA polymerase as it removes single nucleotides from the substrate DNA molecule. The enzymatic activity can be measured with a base-pair resolution. Both ends of the DNA molecule are tethered to its respective optically trapped bead. Read more here: https://lumicks.com/application/dna-replication-optical-tweezers-fluorescence-microscopy/

 

Material supplied

  • T7 DNA polymerase
  • Biotinylated ds pKYB1 DNA with a 25-nt 5′-end overhang
  • dNTP mix
  • Streptavidin coated polystyrene beads (1.76 µm)
  • Polymerase buffer
  • Estimated for 2 weeks of measurements
  • Estimated 1-3 weeks delivery time

Specifications

T7 DNA polymerase

20 µl, 200 Units – 10000 U/ml (typical dilution factor 1:500) T7 DNA polymerase with 8x Histidine tag. In 50% glycerol.

Biotinylated double-stranded pKYB1 DNA

20 µl – 20 ng/µl (typical dilution factor 1:1000) double-stranded expression vector pKYB1 (8393 bp) with a 25-nt 5′-end overhang. Biotin-labeled on the 5′ and 3′ ends of one of the strands, diluted in TE buffer (10 mM Tris HCl pH 8.0 and 1 mM EDTA).

dNTP mix

300 µl – 10 mM of each (typical dilution factor 1:20) deoxynucleotide (dNTP) mix with equimolar concentration dATP, dCTP, dGTP and dTTP. Used by T7 DNA polymerase for incorporating nucleotides.

Polymerase buffer

500 µl – (typical dilution factor 1:20) magnesium based buffer to catalyze the activity of T7 DNA polymerase.

Polystyrene beads (1.76 µm)

25 µl – 1% (w/v) (typical dilution factor 1:1000) streptavidin coated polystyrene microspheres with a diameter of 1.76 µm in water. Binds to biotinylated DNA (e.g., the provided pkYB1) and can be optically manipulated using your optical tweezers system.

Product Resources

Want to learn more?

See our DNA/RNA–Protein Interactions application notes.

Want to use this product in practice?

Find out more about the C-Trap here.