Construction of Recombinant DNA Plasmid for Production 100 Base Pair DNA Ladder for Endless Usage Based on PCR Technique
DOI:
https://doi.org/10.14456/gag.2018.3Keywords:
DNA ladder, synthetic DNA template, Polymerase Chain ReactionAbstract
The standard 100 base pairs (bp) DNA ladder is an essential molecular marker in molecular biology laboratory. This standard DNA marker is normally used to determine the size of the fragments DNA, plasmid and PCR products for gel electrophoresis. Since the standard DNA ladder is one of the major costs of molecular biological research and teaching, therefore, the unlimited in-house production of this DNA ladder could be the effective cost reduction strategy in laboratories. Thus, the purpose of this study is to prepare a DNA ladder template by constructing plasmid DNA inserted with a synthetic 1,500 bp polynucleotides fragment. By using PCR technique, our results showed that the PCR technique able to amplify 11 fragments ranging from 100-1,000 bp and 1,500 bp with high qualification, accuracy in size. Finally, purification of the merged altogether of PCR products showed high quality and quantity of each ladder. Our procedure of in-house for production of 100 bp DNA could be simple, inexpensive, time saving and unlimited production.References
Abbasian M, Seyedi H A, Boroujeni Z K, & Mofid M R (2015) Easy method for production of a home-made DNA ladder in every laboratory. Adv Biomed Res 4: 70. doi: 10.4103/2277-9175.153894
Applichem. (2010 ). Gel Electrophoresis Size Marker, https://www.applichem.com/fileadmin/
Broschueren/Sizemarker.p (June 2017).
Cooney C A, Galbraith J L, & Bradbury E M (1989) A regularly spaced DNA size standard with 10 kbp resolution for pulsed field gel electrophoresis. Nucleic Acids Res 17: 5412.
Natarajan C, Vijayan N N, Vaidyan L K, Mathew A, Srinivas L, & Banerjee M (2008) Universal protocol for generating 100bp size standard for endless usage. Electronic J Biotechnol 11. doi: ARTN 1010.2225/vol11-issue2-fulltext-10
Polyarush S V, Egamberdiev S S, Mansurov D R, & Azimova S S (2003) Preparation of DNA markers based on E-coli plasmid DNA. Chemist Natural Compounds 39: 592-594. doi: Doi 10.1023/
B:Conc.0000018117.85641.B5
Rashno M, Shapouri M R S A, & Jolodar A (2012) Construction of a synthetic vector for preparation of a 100 base pair DNA ladder. Iranian J Biotechnol 10: 106-110.
Riyajan. S, Yentua. W, & Phinchongsakuldit. J (2011) Low Cost DNA Molecular Weight Marker: Primer-Directed Synthesis from pGEM-T Easy Vector. Walailak J Sci & Tech 8: 187-193.
Sekhavati M, Tadayon K, Ghaderi R, Banihashemi R, Jabbari A R, Shokri G, & Karimnasab N (2015) "In-house" production of DNA size marker from a vaccinal Bacillus anthracis strain. Iran J Microbiol 7: 45-49.
Thomas M, & Davis R W (1975) Studies on the cleavage of bacteriophage lambda DNA with EcoRI Restriction endonuclease. J Mol Biol 91: 315-328.
