OPTIMASI METODE LISIS ALKALI UNTUK MENINGKATKAN KONSENTRASI PLASMID
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Abstract
Plasmids are extra chromosomal molecules of DNA that replicate autonomously and found in prokaryote and eukaryote cells. There are a number of methods that are used to isolate plasmids, such as alkaline lysis, boiling lysis, using cesium chloride, and chromatography. Amongst the disadvantages in plasmid isolation methods are lengthy time especially when handling a large number of samples, high cost, and low purity. Alkaline lysis is the most popular for plasmid isolation because of its simplicity, relatively low cost, and reproducibility. This method can be accomplished in 50 minutes to one hour. In this research, the alkaline lysis method was developed to obtain suitable plasmid for applications in a molecular biology laboratory. The aim of this research was to reduce contaminants and improve yield of plasmid. The result of isolation of pICZA plasmid in Escherichia coli gave the concentration of 3.3 to 3.8 µg/µL with the purity of 1.99.
Keywords: Plasmid isolation, pICZ A, Escherichia coli, rapid, alkaline lysisÂ
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ABSTRAK
Plasmid merupakan molekul DNA ekstrakromosomal yang bereplikasi secara mandiri dan ditemukan dalam sel prokariot dan eukariot. Banyak metode yang digunakan untuk isolasi plasmid, seperti: lisis alkali, lisis dengan pemanasan, penggunaan sesium klorida, dan kromatografi. Kelemahan beberapa metode isolasi DNA adalah waktu isolasi yang lama terutama saat isolasi plasmid dalam jumlah banyak, mahal dan kemurniannya yang rendah. Metode lisis alkali merupakan metode yang sangat umum untuk isolasi plasmid karena mudah dilakukan, relatif murah, dan reprodusibilitas. Metode ini dapat dilakukan dalam 50 menit sampai 1 jam. Pada penelitian ini dikembangkan metode lisis alkali untuk memperoleh plasmid yang sesuai untuk penggunaan di laboratorium biologi molekuler. Tujuan dari penelitian ini adalah untuk mengurangi jumlah kontaminan dan meningkatkan konsentrasi plasmid. Hasil isolasi plasmid pICZA dalam Escherichia coli mempunyai konsentrasi antara 3,3 sampai 3,8 µg/µL dan kemurniannya 1,99.
Kata Kunci: Isolasi plasmid, pICZ A, Escherichia coli, cepat, lisis alkali
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References
Birnboim HC, Doli J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513-1523
Brown TA (2010) Gene Cloning and DNA Analysis: An Introduction, ed. Ke-6. Graphicraft Limited. Hongkong.13-17
Dederich DA, Okwuonu G, Garner T, Denn A, Sutton A, Escotto M, Martindale A, Delgado O, Muzny DM, Gibbs RA, Metzker ML (2002) Glass bead purifi-cation of plasmid template DNA for high throughput sequencing of mam-malian genomes. Nucleic Acids Res 30:1-5
Easyselect Pichia Expression Kit for Expression of Recombinant Protein Using pPICZ and pPICZα in Pichia https://tools.thermofisher.com/content/sfs/manuals/easyselect_man.pdf, diakses 20 Oktober 2015
Kiran C, Sreekanth P, Ponnala D, Paithankar KR (2010) A quick and economical method to isolate plasmid DNA for large scale sequencing. Int J Appl Biol Technol 1:1236-1238
Kotchoni SO, Gachomo EW, Betiku E, Shonukan OO (2003) A home made kit for plasmid DNA mini-preparation. Afr J Biotechnol 2:88-90
Palomares LA, Mondaca ST, Ramirez OT (2004) Production of recombinant proteins: challenges and solutions. In: Methods in Molecular Biology: Recom-binant Gene Expression Reviews and Protocols, vol 267. Balbas P & A Lorence (ed). Humana Press Inc., New Jersey
Perez-Ortin JE, Ramon D, Ferrer S, Tordera V (1986) Rapid plasmid isolation, A laboratory experiment. Biochem Edu 14:142-144
Sambrook J, Russell DW (2001) Molecular Cloning, vol. 1.Cold Spring Harbor Laboratory Press., New York
Sato M, Akasaka E, Saitoh I, Ohtsuka M, Nakamura S, Sukarai T, Watanabe S (2012) A simplified protocol for the semi-large scale recovery of plasmids from Escherichia coli grown on agar plates. J Biomed Sci Eng 5:406-408
Yadav P, Yadav A, Garg V, Datta TK, Goswami SL, De S (2011) A novel method of plasmid isolation using laundry detergent. Indi J Exp Biol 49:558-560