Optimizing Different Conditions to Develop a Single Stranded Phage-Lambda DNA (λDNA) Tightrope
International Journal of Biomedical Science and Engineering
Volume 7, Issue 3, September 2019, Pages: 61-67
Received: Aug. 14, 2019;
Accepted: Sep. 9, 2019;
Published: Sep. 24, 2019
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Gali Adamu Ishaku, School of Biological Sciences, University of Essex, Essex, United Kingdom; Department of Biotechnology, Modibbo Adama University of Technology, Yola, Adamawa, Nigeria
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.
Gali Adamu Ishaku,
Optimizing Different Conditions to Develop a Single Stranded Phage-Lambda DNA (λDNA) Tightrope, International Journal of Biomedical Science and Engineering.
Vol. 7, No. 3,
2019, pp. 61-67.
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