Construction of DNA ladder for determination of small size DNA fragments

Vo Thi Thuong Lan; Le Thi Thanh

doi:10.15625/1811-4989/13897

Author affiliations

Authors

Vo Thi Thuong Lan VNU University of Science Hanoi
Le Thi Thanh

DOI:

https://doi.org/10.15625/1811-4989/13897

Keywords:

Từ khóa, Cắt không hoàn toàn với enzyme giới hạn, Plasmid tái tổ hợp, Thang chuẩn DNA 60 bp.

Abstract

DNA marker is commonly used to determine the size of DNA fragments by electrophoresis in
routine molecular biology laboratories. In this study, we report a new procedure to prepare recombinant plasmids pSY-60 which was partially digested by one restriction enzyme for generating DNA markers of 7 fragments from 60 to 420 bp. The procedure included a synthesis of 60 bp DNA fragment with EcoRI sites at both ends using PCR extension, self-ligation of the 60 bp fragments and subcloning the ligated product into plasmid, generating recombinant pSY-60. Once being cloned, 500 ng of 420 bp fragment purified from 100 µL PCR product was incompletely digested by EcoRI, sufficiently producing to 50 acrylamide gels. Our procedure for production of DNA markers could be simple, time saving and inexpensive in comparison with current ones widely used in most laboratories.

Downloads

Download data is not yet available.

References

Tài liệu tham khảo

Abbasian M, Seyedi HE, Boroujeni ZK, Mofd MR (2015) Easy method for production of a home-made DNA ladder in every laboratory. Adv Biomed Res 4:70 - 75.

Abdel-Fattah YR, Gaballa AA (2006) Synthesis of DNA ladder by polymerase chain reaction and optimization of yield using response surface methodology Biotechnology 5: 166–172.

Bauer RJ, Zhelkovsky A, Bilotti K, Crowell LE, Evans TC, McReynolds LA, Lohman GJS (2017) Comparative analysis of the end-joining activity of several DNA ligases. PLoS ONE 12(12): e0190062. https://doi.org/10.1371/journal.pone.0190062.

Chang M, Wang JH, Lee HJ (2008). Laboratory production of 100 base pair DNA molecular weight markers. J. Biochem Biophys Methods 70: 1199–1202.

Cooney CA (1994) Techniques and high resolution DNA size markers for pulsed field gel electrophoresis. Molecular Biotechnology 2:119–127.

Griffiths RA, Barber MD, Johnson PE., Gillbard SM, Haywood MD, Smith CD, Arnold J, Burke T, Urquhart AJ, Gill P (1998) New reference allelic ladders to improve allelic designation in a multiplex STR system. Int Legal Medicine 111: 267-272.

Henrici RC, Pecen T J, Johnston JL, Tan S (2017) The pPSU plasmids for generating DNA molecular weight markers. Scientific Reports 7, Article number: 2438.

Lan VTT, Loan PTT, Duong PAT, Thanh LT, Ha NT, Thuan TB (2012) Straightforward procedure for laboratory production of DNA ladder. Journal of Nucleic Acids, Volume 2012, Article ID 254630, 4 pages. doi:10.1155/2012/254630.

Mehrnejad F, Naderi-Manesh H, Ranjbar B, Maroufi B, Asoodeh A, Doustdar F (2007) PCR-based Gene Synthesis, Molecular Cloning, High Level Expression, Purification, and Characterization of Novel Antimicrobial Peptide, Brevinin-2R, in Escherichia Coli. Appl Biochem Biotechnol 149:109-18. doi: 10.1007/s12010-007-8024-z.

Parker RC, Watson RM, Vinograd J (1977) Mapping of closed circular DNAs by cleavage with restriction endonucleases and calibration by agarose gel electrophoresis. PAS 74: 851–855.

Polyarush SV, Egamberdiev SS, Mansurov DR, Azimova SS (2003). Preparation of DNA markers based on E. coli plasmid DNA. Chem Nat Compounds 39: 592–594.

Wang TY, Guo L, Zhang J (2010) Preparation of DNA ladder based on multiplex PCR technique. Journal of Nucleic Acids, Volume 2010, Article ID 421803, 3 pages, 2010.

Ye C, Gu J, Chen S , Deng A, Li YZ, Li D (2010) Unit cloning and amplification as novel and universal strategies for complex vector construction and small DNA fragment preparation. Electrophoresis 31:2929–2935.