Abstract
λDNA tightrope can be referred to as λDNA is suspended between two beads above the surface of the flow cell which allows the λDNA to be viewed extended rather than in collapse form. It is use for DNA studies such DNA repairs, but when using the normal protocol (at a velocity of 300 µl/minutes, beads size of 5 µm and concentration of 500.0 µg/ml of 1.0 λDNA) it results to multiple tightropes forming on the beads which interference with a many studies. In this study, different conditions were optimized to develop a single stranded λDNA tight rope by taking into consideration the following conditions, velocity (100 to 600), beads size (3 µm and 5µm), concentration (500.0 µg/ml, 375.0 µg/ml and 250.0 µg/ml) and time. To form the tightrope, beads were added into water in an eppendorf tube, centrifuged and sonicated before putting into the flow cell. The flow cell, syringe pump and perfusion tube were all fixed together, where ABC buffer and λDNA were put into the perfusion tube and set according to protocol to make the tightropes. YOYO-1 dye was used to stain the λDNA before using atomic force microscopy to image the tightropes, the same procedure was repeated for the different conditions and all dispense readings of the flow rate were taken at not less than 3.0 µl/minutes. λDNA on the surface of the flow cell was observed at the velocity of 100 and 200 µl/minutes for all the conditions. The optimized result was at velocity of 300µl/ml, concentration 375.0 µg/ml of 1.0 μλDNA and beads size of 3 µl which lasted for 15 minutes which gave the best result of single stranded λDNA tightrope compared to all the conditions and the normal protocol.
Highlights
Phage-Lambda DNA tightrope can be referred to as when λDNA is suspended between two beads above the surface of flow cell which allows the λDNA to be viewed extended rather than in collapse form [1]
The flow cell is first filled with water to check if there is any leakage; beads that have been sonicated at 1 cycle by 80% amplitude were put into the flow cell using a tube with a special tube on it that can fit the tube of the flow cell
To overcome the problems of multiple tightropes, different conditions such as bead size (5 μM and 3 μM), concentration of λDNA (500.0 μg/ml, 375.0 μg/ml and 250.0 μg/ml), velocities of the syringe pump flow rate (100, 200, 300, 400, 500 and 600) and time were used for the optimization
Summary
Phage-Lambda DNA (λDNA) tightrope can be referred to as when λDNA is suspended between two beads above the surface of flow cell which allows the λDNA to be viewed extended rather than in collapse form [1]. The most effective and convincing way to decipher DNA and protein interaction is to observe direct protein binding under well-controlled condition, to actualized this scientist have developed very powerful methods and instruments like Total Internal Reflection Fluorescence Microscopy (TIRFM) and Atomic Force Microscopy (AFM) [3,4,5] One of these well-controlled conditions is through the use of DNA tightrope assay and instrument [1, 6,7]. Optimization studies have been performed to enhance real time imaging of protein-DNA interactions following the single molecule technique which includes DNA-binding protein platforms This approach was initiated over two decades ago when Kabata and colleagues applied dielectrophoresis to extend λDNA [20]. Where optimized result was compared to the normal protocolof forming λDNA (beads size of 5 μM, velocity of 300 μl/Mins. concentration of 500.0 μg/ml at not less than 20 mins)
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